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STUDIES 


THE  SARANAC ■ LABORATORY  FOR  THE 
STUDY  OF  TUBERCULOSIS  /, 


STUDIES    IN    IMMUNITY    TO    TUBERCULOSIS 


I.     Experimental  studies  on  the  cutaneous  reaction  to  tuberculo-protein 
First  Paper:  Factors  governing  the  reaction 

II.     The  anaphylactic  state  in  its  relation  to  resistance  to 

tuberculous  infection  and  tuberculous  disease 

(An  experimental  study) 

III.     Concerning  the  general  tuberculin  reaction 


A.    K.    KRAUSE,    M.D. 


PUBLICATION     OFFICE     OF     THE     JOURNAL     OF     MEDICAL     RESEARCH 
240     LONGWOOD     AVENUE,    BOSTON,    MASS.,    U.S.A. 


J// 

MS 


Studies  in  Immunity  to  Tuberculosis.* 

experimental  studies  on  the  cutaneous  reaction  to 
tuberculo-protein. 

First  Paper. 

FACTORS    GOVERNING   THE    REACTION. 

A.  K.  Krause,  M.D. 
(From  the  Saranac  Laboratory  for  the  Study  of  Tuberculosis.) 

It  is  remarkable  that  the  phenomenon  of  cutaneous  hyper- 
sensitiveness  has  not  attracted  more  of  the  careful  and  earnest 
experimental  work  that  it  deserves.  Hailed  upon  its  dis- 
covery as  of  diagnostic  importance  in  tuberculosis,  the  appli- 
cation of  the  skin  test  upon  human  beings  by  a  multitude  of 
observers  soon  disclosed  that  in  diagnosis  and  prognosis  the 
test  was  of  very  limited  value.  It  is  now  ten  years  since 
von  Pirquet  and  Schick's  first  publication  and  an  enormous 
literature  has  accumulated,  outlining  studies  as  they  have 
been  made  on  skin  hypersensitiveness  on  man.  Out  of  this 
mass  of  contradictory  reports  has  come  comparatively  little 
definite  information,  for  the  reason  that  the  work  was  done 
on  man  under  conditions  that  were  not  easy  of  comparison 
and  control.  It  is  at  once  plain  that  a  study  of  this  biologi- 
cal test  as  correlated  with  anatomical  conditions  could  be 
made  only  rarely  and  in  isolated  instances.  Again,  it  was 
only  in  individual  and  exceptional  cases  that  prolonged 
observation    before  infection,  at  the    time  of  infection,   and 

*  Received  for  publication  June  10,  1916. 


2  KRAUSE. 

during  the  course  of  disease  could  be  carried  out.  As  a 
rule  the  only  clinical  opportunity  was  to  correlate  the  results 
of  skin  tests  with  symptoms  and  physical  signs ;  and  it  is 
notorious  how  mere  symptoms  and  physical  signs  can  lead 
us  astray  when  we  attempt  from  them  to  visualize  what  may 
be  going  on  pathologically  in  a  condition  like  chronic 
phthisis.  However,  observation  on  man  has  contributed 
several  important  facts.  It  has  taught  us  that  a  positive 
cutaneous  test  means  infection  or  the  presence  of  tubercle, 
although  the  disease  tuberculosis  does  not  of  necessity 
exist.  It  has  shown  that  fever  and  intercurrent  infections 
may  on  occasion  blunt  or  abolish  the  capacity  of  the  skin  to 
react.  As  an  index  of  infection  it  has  greatly  advanced  our 
knowledge  concerning  the  epidemiology  of  tuberculosis. 
But  most  of  the  underlying  causes  of  the  skin  reactions  and 
the  reasons  for  their  fluctuations  have  apparently  eluded  the 
clinical  observer. 

It  would  seem  only  natural,  in  view  of  what  I  have  just 
said,  that  students  of  tuberculosis  would  resort  to  animal 
experimentation  to  acquire  data  under  controllable  condi- 
tions. Here  the  infecting  agent  could  be  varied  at  will, 
the  time  of  infection  could  be  definitely  fixed,  the  sensi- 
tiveness of  the  animal  at  any  period  after  infection  could 
be  noted,  and  the  condition  of  the  animal  at  any  time 
could  be  exactly  determined  by  immediate  autopsy.  At 
first  there  was  good  reason  why  animal  experiment  was 
not  fruitful,  for  it  was  found  that  the  skin  of  the  ordinary 
laboratory  animals  that  are  susceptible  to  tuberculosis  {e.g., 
the  guinea-pig  and  the  rabbit)  would  not  react  to  the  v. 
Pirquet  test  (the  method  of  scarification).  Rut  this  obsta- 
cle no  longer  interfered  after  Mendel,1  and  Moussu  and 
Mantoux2  described  their  method  of  the  intracutaneous 
introduction  of  tuberculin  to  which  a  tuberculous  guinea-pig 
will  react  very  sharply. 

Notwithstanding  the  ease  and  applicability  of  the  Moussu- 
Mantoux  method  the  literature  has  remained  singularly  free 
from    experimental   studies   on    skin  sensitiveness.      It    is   no 


STUDIES    IN   IMMUNITY    TO    TUBERCULOSIS.  3 

exaggeration  to  say  that  their  number  can  be  counted  on  the 
fingers  of  one  hand.  Of  these  Romer's3  and  Baldwin's4 
stand  out  most  prominently.  Baldwin  was  concerned  with 
the  conditions  under  which  skin  sensitiveness  might  occur  in 
guinea-pigs.  He  injected  a  number  of  animals  with  various 
products  of  the  tubercle  bacillus  and  of  tuberculous  foci, 
but  could  obtain  no  positive  reactions  unless  he  produced 
tubercles  by  the  inoculation  of  either  living  or  dead  bacilli. 
Therefore,  so  far  as  he  went,  he  concluded  that  only  the 
presence  of  anatomic  tubercle  endows  an  animal  with  the 
capacity  to  react  cutaneously  to  tuberculo-protein.  Romer 
was  interested  more  in  the  meaning  of  the  various  degrees 
of  reactive  power  of  the  animals.  He  concludes  that  the 
intensity  of  the  reaction  varies  directly  with  the  extent  and 
advance  of  the  tuberculous  process. 

As  our  knowledge  of  the  processes  of  immunity  to  tuber- 
culous infection  has  grown,  the  desirability  of  a  better 
insight  into  the  various  phases  of  the  skin  reaction  has 
become  more  and  more  apparent.  At  the  Saranac  Labora- 
tory we  have  been  practising  the  skin  test  on  animals  for 
about  seven  years.  As  time  went  on  I  became  more  and 
more  impressed  with  the  fact  that  at  many  points  the  factors 
that  governed  many  features  of  the  cutaneous  test  approached 
or  resembled  those  that  we  have  come  to  learn  as  being 
associated  with  the  laws  of  immunity  to  tuberculous  infec- 
tion. About  three  years  ago  I  therefore  resolved  to  under- 
take a  more  or  less  exhaustive  study  on  animals  from  several 
points  of  view:  first,  to  study  the  details  of  the  mere 
occurrence  or  non-occurrence  of  the  skin  reaction  (its  rela- 
tion to  time  of  infection  and  extent  of  infection,  its  modifica- 
tion by  other  reactions,  etc.)  ;  and  secondly,  to  study  the 
possible  bearing  that  the  inflammatory  skin  reaction  may 
have  on  immunity  to  cutaneous  re-infection  (see  Romer5  on 
cutaneous  re-infection).  The  present  paper  is  the  first  of  a 
series  in  which  I  hope  to  communicate  the  results  of  these 
studies,  and  shall  deal  as  closely  as  possible  only  with  the 
first  of  the  problems  outlined  above. 


4  KRAUSE. 

A  description  of  the  points  of  resemblance  and  difference 
between  Romer's  method  of  approach  and  my  own  is  here 
necessary. 

The  culture  used  for  infection.  —  In  all  his  experiments 
Romer  inoculated  his  animals  with  a  comparatively  virulent 
strain  of  living  human  or  bovine  bacilli.  He  introduces 
these  in  amounts  varying  from  .1  to  .0000025  milligram,  or, 
when  he  uses  an  emulsion  of  Bovovaccine,  from  .01  to 
.OOOOOOOl  cubic  centimeter.  All  but  a  very  few  of  the 
animals  became  infected  ;  in  several  which  received  exces- 
sively high  dilutions,  he  could  find  no  lesion  at  autopsy. 
Nearly  all  of  his  animals,  too,  if  allowed  to  live  long  enough, 
showed  progressive  tuberculosis.  At  any  given  time  the 
comparative  extent  of  tuberculosis  in  a  given  series  varied, 
depending  on  the  number  of  bacilli  which  the  individuals 
received.  Practically  all  of  his  animals  which  came  to 
autopsy  exhibited  macroscopic  visceral  tuberculosis.  He 
practised  intracutaneous  tests  on  these  animals  at  various 
periods  after  infection  and  studied  the  changing  intensity -of 
reaction. 

In  the  work  which  will  be  presented  in  this  paper  a  study 
was  made,  not  of  the  variations  in  reaction  as  these  occurred 
in  animals  that  had  been  infected  with  different  quantities  of 
virulent  strains,  but  of  the  reactions  induced  in  animals 
infected  by  two  different  strains  of  bacilli  which  produced 
two  very  different  and  easily  distinguished  effects.  I  have 
worked  constantly  with  both  these  strains  of  human  tubercle 
bacilli,  Strain  Ri  and  Strain  H37,  for  seven  years,  and  am 
thoroughly  familiar  with  their  invasiveness  under  many 
different  conditions.  During  all  this  time  their  virulence 
has  remained  fairly  definite  and  constant.  Strain  H37  is  of 
fair  virulence  and  in  the  doses  which  we  employ  will  always 
bring  about  progressive  tuberculosis  in  guinea-pigs,  involv- 
ing the  viscera  and  leading  ultimately  to  the  death  of  the 
animal.  Strain  Ri  will  always  produce  regional  lymphatic 
gland  tuberculosis  after  subcutaneous  inoculation,  but  will 
hardly  ever  cause  gross  visceral  lesions.  Animals  after  Ri 
infection  always  remain  in  good  constitutional  condition  and 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  5 

after  a  while  glands  which  were  once  enlarged  tend  to 
return  to  their  former  size.  Two  years  after  infection  the 
viscera  are  completely  free  from  any  trace  of  tuberculosis 
and  the  regional  glands  are  apparently  almost  normal. 
Microscopically  these  glands  at  this  time  are  often  found  to 
be  completely  sclerotic,  although  in  a  few  animals  I  have 
seen  histological  tubercle  walled  off  by  dense  fibrous  tissue. 
This  is  a  very  different  picture  from  that  which  one  sees  for 
several  months  after  Ri  infection  when  the  regional  glands 
are  usually  partially  caseous.  The  time  at  which  the  Ri 
infection  stops  its  march  along  the  lymphatic  chain  and 
begins  to  retrogress  is  a  point  that  is  very  difficult  to  deter- 
mine with  even  approximate  accuracy.  But  my  experience 
has  taught  me  that  this  change  begins  to  disclose  itself  at 
from  three  or  four  to  six  months  after  infection.  I  judge 
that  this  has  occurred  only  by  paying  attention  to  the  char- 
acter of  the  regional  glands,  when  I  find  that  their  size  is 
diminishing  and  their  consistence  is  beginning  to  be  less 
hard.  It  must  be  remembered  that  in  very  mild,  very  slow, 
and  very  chronic  tuberculosis  changes  occur  with  amazing 
slowness;  they  are  likely  to  be  appreciable  only  after  inter- 
vals of  months.  While  the  Ri  bacillus  always  brings 
about  local  glandular  changes  in  guinea-pigs,  it  has  never  in 
my  observation  produced  lesions  in  them  sufficient  to  cause 
death. 

The  use  of  these  two  different  strains  of  tubercle  bacilli 
provided  the  opportunity  to  study  the  reaction  under  two 
very  distinct  conditions  :  the  one  in  the  presence  of  advanc- 
ing tuberculosis  ;  the  other  in  animals  with  stationary,  self- 
limited,  or  healing  infection.  The  conditions  were  some- 
what simpler  and  more  sharply  differentiated  than  those  in 
Romer's  animals. 

Method  of  infection.  —  Romer  infected  most  of  his  guinea- 
pigs  intraperitoneally  and  some  subcutaneously.  All  of  the 
animals  in  these  experiments  were  infected  subcutaneously, 
so  that  track  could  be  kept  of  the  glandular  changes. 


6  KRAUSE. 

The  antigen  used.  —  Romer  used  old  tuberculin  in  testing 
his  animals.  As  is  well  known  this  is  a  very  complex 
product  consisting  of  a  boiled,  filtered,  condensed,  glyceri- 
nated  extract  of  tubercle  bacilli  and  contains  a  high  propor- 
tion of  peptone  and  common  salt.  He  used  .02  cubic  centi- 
meter of  tuberculin  always  in  a  bulk  of  .1  cubic  centimeter 
of  fluid  (a  dilution  of  I  to  5  in  physiological  salt  solution). 

In  our  own  experiments  we  always  used  a  water  extract 
filtrate  of  dried,  pulverized,  human  (H37)  bacilli,  made  iso- 
tonic by  the  addition  of  common  salt,  and  in  this  work  desig- 
nated as  W.E.  179.  The  dried  residue  of  W.E.  179  weighed 
.02  gram  per  cubic  centimeter  of  extract.  This  W.E.  was 
always  diluted  with  physiological  salt  solution  in  equal  pro- 
portions and  a  total  bulk  of  .2  cubic  centimeter  injected  into 
the  skin.  Every  dose  represented  therefore  .002  gram  of 
dried  substance. 

The  periods  of  testing.  —  In  his  tables  Romer  shows  no 
tests  that  were  made  earlier  than  twenty- one  days  after  infec- 
tion and  none  that  were  continued  more  than  eight  months 
after  infection. 

In  the  following  experiments  tests  were  begun  at  eleven 
days  after  infection,  or  as  soon  as  palpable  changes  in  the 
regional  lymphatic  glands  were  apparent,  and  continued  at 
various  intervals  for  six  hundred  and  sixty-nine  days,  or 
almost  two  years,  after  infection. 

Estimation  of  the  intensity  of  the  reaction.  —  As  is  well 
known  a  reaction  is  positive  when  inflammation  appears 
within  twenty-four  or  forty-eight  hours  at  the  site  of  the 
injection  of  the  tuberculo-protein.  This  inflammation  can 
be  of  all  grades,  from  mere  hyperemia  to  hemorrhagic 
necrosis.  The  scale  of  reaction  that  one  generally  meets 
with  runs  somewhat  as  follows:  Hyperemia;  hyperemia 
and  induration;  hyperemia,  induration,  and  necrotic  center; 
hyperemia,  induration,  and  necrotic-hemorrhagic  center. 
An  atypical  form  of  reaction  is  induration  with  very  slight 
hyperemia    or  induration  with  pallid,   blanched   skin.     Any 


STUDIES    IN   IMMUNITY   TO    TUBERCULOSIS.  7 

marked  hyperemia  after  twenty-four  hours  without  indura- 
tion is  a  rarity.  We  consider  that  the  reacting  area  must 
measure  at  least  ten  millimeters  in  its  longest  diameter  to  be 
noted  as  positive.  To  gain  all  the  information  possible  read- 
ings should  be  made  both  at  twenty-four  and  forty-eight 
hours  after  tuberculin  injection.  And  if  the  reaction  is  of 
the  delayed  type,  not  reaching  its  height  until  after  twenty- 
four  hours  have  elapsed,  a  reading  at  seventy-two  hours  is 
not  superfluous.  As  a  rule  the  twenty-four-hour  reading 
shows  greater  intensity  than  at  forty-eight  hours.  In  the 
following  experiments  the  reactions  will  be  expressed  in 
terms  of  the  extent  of  hyperemia  (redness)  and  induration 
and  of  the  amount  of  central  necrosis  and  hemorrhage,  if 
these  are  present. 

Experiments. 

i.  Infection  of  animals:  On  June  20,  1913.  thirty-one  guinea-pigs 
were  infected  with  living  Ri  tubercle  bacilli  as  follows:  Nos.  1  to  31 
received  subcutaneous  inoculations  in  the  right  groin,  each  .1  cubic  centi- 
meter of  a  centrifugalized  emulsion. 

On  June  28,  1913,  thirty-one  guinea-pigs  were  infected  with  living  H37 
tubercle  bacilli  as  follows :  Nos.  32  to  62  received  subcutaneous  inocula- 
tions in  the  right  groin,  each  .1  cubic  centimeter  of  a  centrifugalized 
emulsion. 

The  Ri  emulsion  was  a  heavier  emulsion  than  the  H37,  so  the  Ri 
animals  were  inoculated  with  greater  numbers  of  bacilli,  but  of  a  low 
virulent  type. 

2.     Intracutaneous  tests  :     Eleven  days  after  infection  : 

On  Ri  animals:  July  I,  1913  :  Nos.  1  to  44  injected  intracutaneously, 
each  with  .2  cubic  centimeter  diluted  W.E.  179  (1  cc.  =  .01  gm  .). 

July  2  (24  hours)  :  No  reaction  in  any ;  Superficial  inguinal  glands  are 
apparently  normal  in  all. 

July  3  (48  hours)  :     No  reactions. 

On  H37  animals:  July  9,  1913:  The  glands  of  Nos.  32,  33,  and  34 
are  apparently  slightly  enlarged ;  those  of  35  are  normal ;  Nos.  32  to  35 
injected  intracutaneously,  each  with  .2  cubic  centimeter  diluted  W.E.  179 
(1  cc.  =  .01  gm.). 

July  11  (48  hours):  No.  32,  Redness  and  induration,  25  x  25  milli- 
meters; Necrotic  center,  6  x  6  millimeters.  No.  33,  Questionable.  No. 
34,  Negative.     No.  35,  Negative. 


8  KRAUSE. 

Eleven  days  after  infection,  when  no  anatomic  changes  are 
palpable  in  the  Ri  animals  and  slight  changes  have  appar- 
ently developed  in  the  H37  animals,  the  Ri  animals  are  still 
insensitive,  while  the  H37  animals  are  becoming  sensitive. 
Cutaneous  hypersensitiveness  therefore  apparently  begins  to 
develop  with  the  development  of  tubercle. 

3.     Intracutaneous  tests:     Eighteen  days  after  infection: 

On  Ri  animals  :  July  8,  1913  :  Injected  Nos.  1  to  8  intracutaneously  in 
the  usual  way. 

July  9  (24  hours)  :  No.  1,  Negative;  Gland,  slightly  +.  No.  2,  Ques- 
tionable; Gland,  normal.  No.  3,  Redness  and  induration,  20  x  20  milli- 
meters; Gland,  normal.  No.  4,  Redness,  12  x  12  millimeters ;  Gland, 
normal.  No.  5,  Slight  redness  and  induration,  15x15  millimeters; 
Gland,  +.  No.  6,  Questionable;  Gland,  O.  No.  7,  Questionable; 
Gland,  O.  No.  8,  Induration  and  pale  pink  areola,  25  x  20  millimeters; 
Necrotic  center,  3  x  3  millimeters ;  Gland,  definitely  +. 

At  eighteen  days  sensitiveness  and  glandular  changes  are  beginning  in 
the  Ri  animals. 

On  H37  animals:  July  16,  1913  :  Injected  Nos.  32  to  39  intracutane- 
ously in  the  usual  way. 

July  17  (24  hours):  No  32,  Hemorrhagic  center,  7x7  millimeters; 
Area  of  redness,  30  x  25  millimeters ;  Induration,  +  +  + ;  Gland,  +  +. 
No  ^^,  Redness  and  induration,  30x25;  Induration,  -\ — (- ;  Glands, 
+  H — K  No.  34,  Redness  and  induation,  30  x  20,  pale  center;  Indura- 
tion, +  +;  Glands,  -f- +.  No.  35,  Redness  and  induration,  20x20; 
Induration,  -+-  -j-;  Glands,  +  + +.  No.  36,  Redness  and  induration, 
30  x  20;  Induration,  +  +;  Glands,  +  +.  No  37,  Redness  and  indura- 
tion, 30  x  30 ;  Hemorrhagic  center,  5x5;  Induration,  +  +;  Glands,  -f-. 
No.  38,  Redness  and  induration,  25x25;  Induration,  ++;  Glands, 
-) — K  No.  39,  Redness  and  induration,  25x20;  Induration,  -\ — hi 
Glands,  slightly  +• 

July  18  (48  hours)  :  No.  32,  Necrotic  center,  7x7]  Redness,  30  x  25  ; 
Induration,  +  +  +•  No.  ^3^  Redness  and  induration,  15x15;  Indura- 
tion, H — (-•  No.  34,  Redness  and  induration,  25  x  15  ;  Induration,  -\ — (-  ; 
Necrotic  center,  2x2.  No.  35,  Redness  and  induration,  15x15;  Indu- 
ration, + +.  No.  36,  Redness  and  induration,  20x20;  Induration, 
+  -f-.  No.  37,  Redness  and  induration.  30  x  20  ;  Necrotic  center,  3  x  ^^ 
Induration,  -\ — K  No.  38,  Redness  and  induration,  20  x  15  ;  Induration, 
+  +.     No.  39,  Redness  and  induration,  20  x  15  ;  Induration,  +  +. 

At  eighteen  days  the  H37  animals  are  uniformly  sensitive, 
and  much  more  so  than  the  Ri  guinea-pigs.  The  glandular 
reaction   of  the   former  to   infection   is   much   more    marked 


STUDIES    IN   IMMUNITY   TO   TUBERCULOSIS.  9 

than  that  in  the  latter.  From  these  results  there  are  already 
suggestions  that  the  intensity  of  the  reaction  runs  parallel 
with  the  amount  of  tuberculous  involvement  and  conse- 
quently with  the  amount  of  absorption  from  the  foci  of 
disease. 

From  this  time  on,  once  the  establishment  of  infection  and 
disease  in  both  lots  of  animals  was  assured,  the'  tests  were 
not  always  made  on  both  Ri  and  H37  animals  at  exactly  the 
same  intervals  after  infection.  Hundreds  of  inoculations  on 
guinea-pigs  with  H37  had  already  proved  for  us  that  the 
course  of  H37  infection  is  very  regular.  After  subcutaneous 
inoculation  the  spleen  generally  becomes  involved  about  the 
fourth  week,  the  lungs  and  liver  about  the  fifth  and  sixth 
weeks,  and  death  usually  follows  from  the  sixth  week  on. 
There  was  no  reason  to  believe  that  the  results  of  infection 
would  be  any  different  in  these  animals. 

4.     Intracutaneous  tests :     Twenty-five  days  after  inoculation  : 

On  Ri  animals:  July  15,  1913  :  Injected  Nos.  1  to  12  intracutaneously 
in  the  usual  way. 

July  16  (24  hours)  :  No.  1,  Negative  ;  Gland,  -f-.  No.  2,  Redness  and 
induration,  15x15;  Induration,  slight ;  Central  necrosis  and  hemorrhage, 
4x4;  Gland,  ?.  No.  3,  Redness  (pale),  30x25  ;  Induration,-!-;  Gland. 
+  .  No.  4,  Redness  (pale),  20 x  15  ;  Induration,  slight ;  Gland, -(-•  No  5, 
Redness,  25  x  20,  pale  center;  Induration,  slight;  Gland,  -| — (-•  No.  6, 
Redness,  25  x  20,  pale  center ;  Induration, +;  Gland,  +  +.  No.  7,  Red- 
ness (pale),  30  x  15  ;  Induration,  + ;  Gland, +.  No.  8,  Redness,  20  x  20, 
pale  center;  Induration,  + ;  Gland, +.  No.  9,  Redness,  25  x  20,  pale 
center;  Induration,  -f- ;  Gland,  +.  No.  10,  Pallid  reaction,  15  x  15; 
Induration,  + ;  Gland,  +  +.  No.  11,  Necrotic  hemorrhagic  center, 
4x4;  Redness,  25x25;  Induration,  +;  Glands,  +•  No.  12,  Redness, 
25  x  20  ;  Induration,  -f-  + ;  Glands,  +  +. 

July  17  (48  hours)  :  No.  1,  Negative.  No.  2,  Central  necrosis,  6x6; 
Redness,  10  x  10;  Induration,  -(-.  No.  3,  Redness  and  induration, 
15x15;  not  so  pale  this  morning.  No.  4,  Almost  completely  faded. 
No  5,  Redness  and  induration,  15  x  15.  No  6,  Redness  and  induration, 
15x15.  No.  7,  Almost  completely  faded;  Slight  induration.  No.  8, 
Redness  and  induration,  15  x  15.  No.  9,  Necrotic  center,  2x2;  Red- 
ness, 20  x  20  ;  Induration,  +.  No.  10,  Necrotic  center,  7  xj  ;  Redness, 
10  x  10;  Induration,  +.  No.  11,  Necrotic  center,  4x4;  Redness, 
10  x  10;  Induration,  +.  No.  12,  Necrotic  center,  5x5;  Redness, 
10  x  10;    Induration,  +. 


IO  KRAUSE. 

At  twenty-five  days  the  reactions  in  the  Ri  animals  are 
increasing  in  intensity,  but  are  not  so  marked  as  those  in  the 
H37  animals  eighteen  days  after  infection. 

5.  Intracutaneous  tests :     Thirty  days  after  infection  ; 

On  H37  animals:  July  28,  1913  :  Injected  Nos.  32  to  39  intracutane- 
ous^ in  the  usual  way. 

July  29  (24  hours)  :  No.  32,  Redness  and  induration,  40  x  30;  Hem- 
orrhagic center,  20  x  15  ;  Induration,  +  +  -f- ;  Glands,  +  +  +.  No.  23, 
Redness  and  induration,  40  x  30 ;  Hemorrhagic  center,  5x5;  Induration, 
+  +  +;  Glands,  +  -\ — K  No.  34,  Redness  and  induration,  30  x  30  ; 
Induration,  +  -j-;  Glands,  +  +.  No.  35,  Redness  and  induration,  35  x 
40 ;  Induration,  -f  +  + ;  Glands,  ++-(-.  No.  36,  Redness  and  indura- 
tion, 20  x  25  ;  Induration,  +  +  +  ;  Hemorrhagic  center,  5x5;  Glands, 
+  +  +•  No.  37,  Redness  and  induration,  35  x  35  ;  Induration,  -f-  +  +  ; 
Hemorrhagic  center,  5x5;  Glands,  +  +  +.  No  38,  Redness  and 
induration,  30  x  30  (pale)  ;  Induration,  -f-  + ;  Hemorrhagic  center,  3x3; 
Glands,  -\ — (-  +.  No.  39,  Redness  and  induration,  20  x  20  (pale)  ; 
Induration,  -\ — (-  ;  Hemorrhagic  center,  3  x  3  ;  Glands,  +. 

July  30  (48  hours):  No.  32,  Redness  and  induration,  30x30; 
Hemorrhagic,  necrotic  center,  20  x  20.  No.  33,  Redness  and  induration, 
25  x  20;  Necrotic  center,  5x5.  No.  34,  Redness  and  induration,  25  X20  ; 
Necrotic  center,  5x5.  No.  35,  Redness  and  induration,  20  x  20 ;  Ne- 
crotic center,  5x5.  No.  36,  Redness  and  induration,  15  x  15;  Necrotic 
center,  5x5.  No.  37,  Redness  and  induration,  35  x  35  ;  Necrotic  center, 
ro  x  10.  No.  38,  Redness  and  induration,  30x30;  Necrotic  center, 
5x5.     No.  39,  Redness  and  induration,  20  x  20;  Necrotic  center,  5x5. 

With  the  development  and  extension  of  the  disease  hyper- 
sensitiveness  is  increasing. 

6.  Intracutaneous  tests :     Thirty-eight  days  after  infection: 

On  Ri  animals:  July  28,  1913 :  Injected  Nos.  1  to  16  intracuta- 
neous^ in  the  usual  way. 

July  29  (24  hours)  :  No.  1,  Redness  and  induration,  20  x  20 ;  Hemor- 
rhagic center,  5x5;  Glands,  -f-  +.  No.  2,  Redness  and  induration, 
30x20;  Hemorrhagic  center,  3x3;  Glands, -j-.  No.  3,  Redness  and 
induration,  30  x  20 ;  Induration,  -f-  +  ;  Pale  center,  15  x  15  ;  Glands,  +• 
No.  4,  Redness  and  induration,  25  x  25  ;  Pale  center,  5x5;  Glands,  +■ 
No.  5,  Redness  and  induration,  25  x  25  ;  Induration,  -\ — (-  ;  Glands,  -\ — (-• 
No.  6,  Redness  and  induration,  25x30;  Induration,  ++;  Glands, 
-j-.  No.  7,  Redness  and  induration,  20  x  20 ;  Glands,  -(-•  No.  8,  Red- 
ness and  induration,  25  x  25  ;  Induration,  +  +  ;  Hemorrhagic  center, 
5x5;  Glands,  +  +  +•  No.  9,  Redness  and  induration,  25  x  30;  Hem- 
orrhagic center,  8x8;  Induration,  -\ — | — (-;  Glands,  slightly  +.  No.  10, 
Redness  and  induration,  25  x  25  ;  Hemorrhagic  center,  5x5;  Induration, 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  I  I 

+  +  +;  Glands,  +  +.  No.  n,  Redness  and  induration,  30x35; 
Hemorrhagic  center,  10  x  10;  Induration,  +  +  +;  Glands,  +.  No.  12, 
Redness  and  induration,  30  x  25  ;  Hemorrhagic  center,  10  x  10;  Indura- 
tion, +  +;  Glands,  + +.  No  13,  Redness  and  induration,  25x20; 
Induration,  +  ;  Glands,  +.  No.  14,  Redness  and  induration,  30  x  25  ; 
Induration,  +;  Glands,  +.•  No.  15,  Redness  and  induration,  30x30; 
Induration,  +  -f- ;  Glands,  +  +•  No.  16,  Redness  and  induration, 
30  x  35  ;  Induration,  +  +  ;  Glands,  +  +. 

July  30  (48  hours)  :  No.  1,  Redness  and  induration,  15  x  15  ;  Necrotic 
center,  3x3.  No.  2,  Faded;  Redness  and  induration,  8x8.  No.  3, 
Redness  and  induration,  20  x  25  ;  Necrotic  center,  3x3.  No.  4,  Redness 
and  induration,  15x15;  Necrotic  center,  3x3.  No.  5,  Redness  and 
induration,  15  x  15  ;  Necrotic  center,  3x3.  No.  6,  Redness  and  indura- 
tion, 15  x  15  ;  Necrotic  center,  5x5.  No.  7,  Slight  redness  and  indura- 
tion; Faded.  No.  8,  Redness  and  induration,  20  x  20;  Necrotic  center, 
5  x  5.  No.  9,  Redness  and  induration,  15  x  20;  Necrotic  center,  2x2. 
No.  10,  Redness  and  induration,  20  x  20  ;  Necrotic  center,  5x5.  No.  1 1 , 
Redness  and  induration,  25  x  30 ;  Necrotic  center,  3x3.  No.  12,  Red- 
ness and  induration,  20  x  20;  Necrotic  center,  7x7.  No.  13,  Redness 
and  induration,  15  x  15  ;  Necrotic  center,  4x4.  No.  14,  Faded;  Slight 
redness  and  induration.  No.  15,  Redness  and  induration,  20x20; 
Necrotic  center,  4x5.  No.  16,  Redness  and  induration,  25  x  20  ;  Necrotic 
center,  2x2. 

It  will  be  seen  that  the  reactions  in  the  Ri  animals  are 
steadily  increasing  in  severity  and  at  thirty-eight  days 
resemble  those  noted  in  the  H37  animals  at  thirty  days. 
They  should  be  compared  with  the  following  results  obtained 
on  H37  guinea-pigs,  also  at  thirty-eight  days: 

On  H37  animals:  August  5,  1913  :  Nos.  40  to  43  injected  intracuta- 
neous^ in  the  usual  way. 

August  6  (24  hours)  :  No.  40,  Redness  and  induration,  40  x  30 ; 
Induration,  +  +  +;  Hemorrhagic  necrotic  center,  10x10;  Glands, 
+  -+-;  Ulcerated  at  site  of  infection.  No.  41,  Redness  and  induration, 
40x40;  Induration,  +  +  +;  Hemorrhagic  necrotic  center,  15x15; 
Glands,  + +.  No.  42,  Redness  and  induration.  40x40;  Induration, 
+  +  +  ;  Hemorrhagic  necrotic  center,  10  x  10  ;  Glands,  +  +  ;  Ulcerated 
at  site  of  infection.  No.  43,  Redness  and  induration,  40x40;  Indura- 
tion, +  +  +;  Hemorrhagic  necrotic  center,   15  x  15;  Glands,  +  +  +• 

August  7  (48  hours)  :  No.  40,  Redness  and  induration,  30  x  30 ; 
Induration,  +  +  + ;  Necrosis,  10x10.  No.  41,  Redness  and  indura- 
tion, 35  x  35  ;  Induration,  +  +  +;  Necrosis,  15  x  15.  No.  42,  Redness 
and  induration,  25x25;  Induration,  +  +  +;  Necrosis,  15x15.  No. 
43,  Redness  and  induration,  20x20;  Induration,  +  + ;  Necrosis, 
10  x  10. 


12  KRAUSE. 

A  glance  at  the  above  results  shows  at  once  that  the 
reactions  in  the  guinea-pigs  suffering  from  progressive 
tuberculosis  are  still  increasing  in  severity.  They  are  much 
more  intense  than  those  in  the  Ri  animals  at  the  same  time. 
From  this  time  on  the  Ri  guinea-pigs  were  not  tested  until 
the  one  hundred  and  twenty-third  day  after  infection,  and 
were  subsequently  tested  at  intervals  until  almost  two  years 
had  elapsed  since  their  infection.  It  was  thought  wisest  not 
to  test  them  too  often,  but  to  conserve  them  and  study  the 
reactions  while  healing  and  arrest  of  the  tuberculous  process 
were  going  on.  Before  they  were  killed  the  H37  animals 
were  tested  once  more,  namely,  on  the  forty-sixth  day  after 
infection,  as  follows: 

7.     Intracutaneous  tests:     Forty-six  days  after  infection  : 

On  H37  animals:  August  13,  1913  :  Nos.  44  to  62  were  injected 
intracutaneous!)'  in  the  usual  way. 

August  14  (24  hours):  No.  44,  Redness  and  induration,  35x35; 
Induration,  +  -j — \-\  Hemorrhagic  necrosis,  15x15;  Glands,  -+-■  No. 
45,  Pallid  type  of  reaction,  25x25;  Induration,  + ;  Glands,  -f- +. 
No.  46,  Redness  and  induration,  30x30;  Induration,  -| — \-\  Hemor- 
rhagic center,  15x10.  No.  47,  Redness  and  induration,  35x30; 
Induration,  -| — I — f-  ;  Glands,  +  +  +.  No.  48,  Redness  and  induration, 
50  x  40 ;  Induration,  +  -f-  -f-  +  ;  Glands,  +  +  +  +.  No.  49,  Redness 
and  induration,  35x30;  Induration,  +  H — h;  Hemorrhagic  center, 
10x10;  Glands,  H — | — K  No.  50,  Redness  and  induration,  35x35; 
Induration,  -\ — (-  -f- ;  Hemorrhagic  center,  20x10;  Glands,  +  + +• 
No  51,  Redness  and  induration,  35x35;  Induration,  -f-  4- ;  Hemor- 
rhagic center,  15  x  10;  Glands,  -)--)-.  No.  52,  Redness  and  induration, 
30x30;  Induration,  +;  Glands,  +  +.  No.  53,  Pallid  type,  20x20; 
Induration,  slight;  Hemorrhagic  center,  10x10;  Glands,  +  + +• 
No.  54,  Redness  and  induration,  35  x  30;  Induration,  +  +  +;  Hemor- 
rhagic center,  20x20;  Glands,  O.  No.  55,  Redness  and  induration, 
30x30;  Induration,  +  +;  Glands,  +  -\ — K  No.  56,  Pallid  type, 
25  x  25  ;  Induration,  H — h|  Hemorrhagic  center,  10  x  10;  Glands,  -f-  +• 
No.  57,  Redness  and  induration,  30  x  30 ;  Induration,  -J-  +  +;  Glands, 
+  +  -(-•  No.  58,  Redness  and  induration,  30  x  30 ;  Induration,  +  -f-  -f-  ; 
Hemorrhagic  center,  10  x  10;  Glands,  -\ — \-  -j-.  No.  59,  Redness  and 
induration,  30  x  30 ;  Induration,  +  -f-  -f- ;  Glands,  +  H — I — K  No.  60, 
Redness  and  induration,  25  x  25  ;  Induration,  -| — (-  +  ;  Hemorrhagic 
center,  10x10;  Glands,  -\ — | — (-•  No.  6r,  Redness  and  induration, 
25x25;  Induration,  -)-  -| — (-;  Glands,  -) — | — (-■  No.  62,  Redness  and 
induration,  25  x  25  ;  Induration,  +  -f-;  Glands,  +. 

August    15   (48  hours):     No.    44,    Redness   and    induration,  25x25; 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  1 3 

Induration,  +  +  +;  Necrosis,  15  x  15.  No.  45,  Redness  and  indura- 
tion, 20  x  15  ;  Induration,  -f- ;  Fading.  No.  46,  Redness  and  induration, 
25x25;  Induration,  -f  +  + ;  Hemorrhagic  necrosis,  15  x  10.  No.  47, 
Redness  and  induration,  25  x  20 ;  Induration,  +  +  + ;  Necrosis,  7x7. 
No.  48,  Redness  and  induration,  25  x  20 ;  Induration,  +  +  ;  Necrosis, 
5x5.  No.  49,  Redness  and  induration,  25  x  25  ;  Induration,  +  +  + ; 
Necrosis,  15x15.  No.  50,  Redness  and  induration,  30x20;  Indura- 
tion, +  +  +;  Necrosis,  20x20.  No.  51,  Redness  and  induration, 
25x20;  Induration,  ++;  Necrosis,  10x10.  No.  52,  Redness  and 
induration,  25x25;  Induration,  +  +;  Necrosis,  10x10.  No.  53, 
Redness  and  induration,  25  x  20 ;  Induration,  -\ — |- ;  Hemorrhagic 
necrosis,  10  x  10.  No.  54,  Redness  and  induration,  25  x  25  ;  Induration, 
+  +;  Necrosis,  10x10.  No.  55,  Redness  and  induration,  25x25; 
Induration,  -\ — h  ;  Necrosis,  10  x  10.  No.  56,  Redness  and  induration, 
25x30;  Induration,  + -| — (-;  Scab,  5x10.  No.  57,  Redness  and 
induration,  25  x  25  ;  Induration,  +  +  +.  No.  58,  Redness  and  indura- 
tion, 30  x  20  ;  Induration,  -| — | — (-  ;  Necrosis,  20  x  10.  No.  59,  Redness 
and  induration,  20  x  20  ;  Induration,  -\ — (-  -f- ;  Necrosis,  12  x  12.  No. 
60,  Redness  and  induration,  25  x  25  ;  Induration,  -\ — f-  +  ;  Hemorrhagic 
necrosis,  10  x  10.  No.  61,  Redness  and  induration,  20  x  20  ;  Induration, 
H — h;  Necrosis,  5x5.  No.  62,  Redness  and  induration,  25x25; 
Induration,  -\ — | — (-• 

These  were  the  last  tests  that  were  made  on  members  of 
the  H37  series  —  those  with  progressive  tuberculosis.  To 
summarize:  they  displayed  increasing  cutaneous  hyper- 
sensitiveness  from  eleven  days  after  infection,  when  the  first 
regional  glandular  changes  were  noted,  up  to  forty-six  days, 
when  the  disease  was  well  advanced.  At  every  stage,  too, 
their  reactions  were  more  intense  than  those  in  guinea-pigs 
suffering  from  Ri,  self-limited  tuberculosis.  On  August  16 
and  17  autopsies  were  performed  on  various  individuals  of 
the  H37  group,  and  advanced  tuberculosis  of  the  viscera, 
lungs,  livers,  and  spleens  was  found  in  all. 

8.  Intracutaneous  tests :  One  hundred  and  twenty-three  days  after  infec- 
tion : 

On  Ri  animals  :  October  21,  1913  :  Injected  Nos.  1  to  8  intracuta- 
neously  in  the  usual  way. 

October  22  (24  hours):  No.  1,  Redness  and  induration,  20  x  20 ; 
Induration,  -| — (- ;  Glands,  -\ — K  No.  2,  Pallid  type;  Induration, 
15  x  15  ;  Induration,  -f- ;  Glands,  +  -f-.  No.  3,  Redness  and  induration, 
30  x  25  ;  Induration,  -\ — (- ;  Glands,  +.  No.  4,  Redness  and  indura- 
tion, 15  x  15;    Induration,  -| — (- ;    Glands,  -\ — (-■    No.   5,    Redness   and 


14  KRAUSE. 

induration,  20  x  20;  Induration,  ++;  Glands,  + +•  No.  6,  Redness 
and  induration,  30  x  20;  Induration,  -\ — h ;  Glands,  -\ — (-■  No.  7,  Red- 
ness and  induration,  30  x  20 ;  Induration,  -\ — (- ;  Glands,  +.  No.  8, 
Redness  and  induration,  30  x  30;  Induration,  -f-  +;  Glands,  -\ — I — K 

October  23  (48  hours)  :  No.  1,  Subsiding  ;  Red  papule,  8x8.  No.  2, 
Subsiding;  Red  papule,  10 x  10.  No.  3,  Redness  and  induration,  15  x  15. 
No.  4,  Subsiding;  Red  papule,  8x8.  No.  5,  Redness  and  induration, 
25  x  25.  No.  6,  Red  papule,  10  x  10.  No.  7,  Subsiding;  Small  papule. 
No.  8,  Red  papule,  12  x  12. 

The  reactions  are  distinctly  less  intense  than  in  the  same 
group  two  months  ago.  The  disease  is  evidently  retrogress- 
ing.    On  October  23  autopsies  were  performed   on  Nos.  1  to 

8.  No  animal  showed  changes  that  extended  farther  than 
the  retroperitoneal  glands.  As  a  rule  the  regional  glands  are 
not  opaque:  they  are  slightly  to  moderately  enlarged,  but 
none  is  caseous.     There  is  no  trace  of  visceral  disease. 

9.  Intracutaneous  tests :     One  hundred  and  sixty-three  days  after  infec- 

tion : 

On  Ri  animals:  December  1,  1913:  Injected  Nos.  9  to  16  intrai_u- 
taneously  in  the  usual  way. 

December  2  (24  hours)  :  No.  9,  Redness  and  induration,  30  x  30 ; 
Induration,  -+-  +  +•  No.  10,  Redness  and  induration,  20  x  20 ;  Indura- 
tion, slight.  No.  11,  Redness  and  induration,  30  x  25 ;  Induration, 
+  +  +•  No.  12,  Redness  and  induration,  30  x  30;  Induration,  +  +  +• 
No.  13,  Redness  and  induration,  30  x  20 ;  Induration,  +  +.  No.  14, 
Redness  and  induration,  30  x  30;  Induration,  +  +  +.  No.  15,  Redness 
and  induration,  30  x  20 ;  Induration,  +  +  +■  No.  16,  Redness  and 
induration,  20  x  20  ;  Induration,  +• 

December  3  (48  hours) :  No.  9,  Redness  and  induration,  20  x  20 ; 
Induration,  +  +•  No.  10,  Considerably  faded;  Slight  redness  and  indu- 
ration. No.  11,  Considerably  faded;  Slight  redness  and  induration, 
10  x  10.  No.  12,  Considerably  faded;  Slight  redness  and  induration, 
10  x  10.  No.  13,  Considerably  faded;  Slight  redness  and  induration, 
10x10;  Slight  necrosis.  No.  14,  Like  No.  13.  No.  15,  Like  No.  13. 
No.  16,  Redness  and  induration,  20  x  15  ;  Induration,  +  -J-. 

On  December  3  autopsies  were  performed  on  Nos.  9  to 
16.  The  lesions  are  about  the  same  in  all.  There  is  no  vis- 
ceral disease.  All  animals  are  very  fat.  The  regional  glands 
are  enlarged  and  are  firmer  than  normal.  It  is  noteworthy 
that  as  the  disease  heals  the  reaction  tends  to  fade  more 
rapidly. 


STUDIES   IN    IMMUNITY   TO   TUBERCULOSIS.  1 5 

10.  Intracutaneous  tests  :     Two  hundred  and  fifty-six  days  after  infection : 

On  Ri  animals:  March  3,  1914:  Injected  Nos.  17  to  20  intracu- 
taneously  in  the  usual  way. 

March  4  (24  hours)  :  No.  17,  Slight  induration  and  redness,  12  x  12  ; 
Glands,  +  and  rather  hard.  No.  18,  Pale  red  area,  20  x  20  ;  Induration 
very  slight;  Glands,  +  and  hard.  No.  19,  Induration,  +;  Redness  not 
marked;  Glands,  O.  No.  20,  Pale  red  area,  20  x  20;  Induration,  +  +; 
Glands,  -f-  +  ,  hard. 

March  5  (48  hours)  :  No.  17,  Faded;  No  longer  positive.  No.  18, 
Slight  induration.  No.  19,  Faded;  Slight  induration.  No.  20,  Redness 
and  induration,  15x15;  Induration,  -| — (-• 

11.  Intracutaneous  tests;     Two    hundred    and  eighty-four    days   after 

infection : 

On  Ri  animals:  April  1,  1914:  Nos.  17  to  23  were  given  intracu- 
taneous tests  with  .2  cubic  centimeter  diluted  W.E.  179  (1  cubic  centi- 
meter =  .01  gram.).  At  the  same  time  they  were  inoculated  intracu- 
taneous^ with  living  H37  and  Ri  bacilli  on  the  opposite  side  of  the  body. 
Only  a  record  of  the  intracutaneous  tests  will  be  given  here. 

April  2  (24  hours)  :  No.  17,  Redness  and  induration,  30  x  20.  No. 
18,  Redness  and  induration,  35  x  35.  No.  19,  Redness  and  induration, 
25x25.  No.  20,  Redness  and  induration,  25x25.  No.  21,  Redness 
and  induration,  20  x  20.  No.  22,  Redness  and  induration,  10  x  10.  No. 
23,   Redness  and  induration,  25  x  20. 

April  3  (48  hours)  :  No.  17,  Redness  and  induration,  15  x  15.  No.  18, 
Redness  and  induration,  20  x  20.  No.  19,  No  induration ;  Redness, 
15  x  15.  No.  20,  Redness  and  induration,  15  x  15.  No.  21,  Redness 
and  induration,  10  x  10.  No.  22,  Redness  and  induration,  15  x  15.  No. 
23,  Redness  and  induration,  20  x  20. 

It  will  be  remembered  that  Nos.  1 7  to  20  were  tested 
twenty-eight  days  before,  on  March  3.  They  now  reacted 
better  than  they  did  then  and,  in  general,  better  than  Nos. 
21  to  23,  which  have  not  been  tested  until  now. 

12.  Intracutaneous  tests :     Four  hundred   and   twenty-eight   days   after 

infection  : 

On  Ri  animals:  August  21,  1914:  Injected  Nos.  25  to  27  intracu- 
taneous^ in  the  usual  way  and  at  the  same  time  with  virulent  living  H37 
bacilli  in  the  skin  of  the  opposite  side. 

August  22  (24  hours)  :  No.  25,  Redness  and  induration,  both  slight; 
Area  measures  25  x  20.  No.  26,  Pallid  reaction,  15  x  15.  No.  27,  Slight 
redness  and  induration,  20  x  20. 


1 6  KRAUSE. 

13.  Intractdaneous  tests:  Six  hundred  and  sixty-nine  days  after  infec- 
tion: 

On  Ri  animals:  April  20,  1915  :  Injected  Nos.  28,  30,  and  31  intra- 
cutaneous^ in  the  usual  manner  and  at  the  same  time  with  living  virulent 
H37  bacilli  in  the  skin  of  the  opposite  side. 

April  21  (24  hours)  :  No.  28,  Pale  pink  and  indurated,  20  x  20;  Indu- 
ration, +  +.  No.  30,  Pale  pink  and  slightly  indurated,  30x20.  No. 
31,  Redness  and  induration,  -j — (-»  20  x  20. 

April  22  (48  hours)  :  No.  28,  Redness  and  induration,  25  x  25  ;  Indu- 
ration, +  +  +.  No.  30,  Pink,  10  x  10;  Induration  very  slight.  No.  31, 
Redness  and  induration,  15  x  15  ;  Induration,  -\ — |— 

April  23  (72  hours)  :  No.  28,  Redness  and  induration,  20  x  20 ;  Indu- 
ration, +  +  +.  No.  30,  Subsided.  No.  31,  Redness  and  induration, 
10  x  10;  Induration,  -| — (-• 

Discussion.  —  The  skin  reactions  have  herein  been  studied 
in  two  very  different  lots  of  tuberculous  guinea-pigs:  in 
one  series,  the  Ri  animals,  which  were  suffering  from  a  self- 
limited  tuberculosis  that  after  a  while  showed  a  tendency  to 
heal;  in  another  series,  the  H37  animals,  which  had  pro- 
gressive tuberculosis  which  ultimately  would  have  caused  the 
death  of  the  animals.  In  both  series  the  skin  reaction . 
became  positive  with  the  palpable  establishment  of  tubercle : 
at  eleven  days  in  the  H37  animals;  at  about  eighteen  days 
in  the  Ri  animals.  In  those  with  progressive  disease  the 
reactions  at  any  given  time  were  always  more  intense  than  in 
those  with  disease  well  under  control.  In  the  animals  with 
self-limited  disease  the  vigor  of  the  reaction  followed  the 
course  of  the  disease.  As  disease  developed,  the  reaction 
increased  in  severity.  After  it  had  reached  a  standstill  and 
began  to  heal,  the  reaction  became  milder.  The  ability  to 
react  never  entirely  disappeared,  and  this  raises  the  question 
as  to  whether  an  infection  like  tuberculosis,  once  it  gains 
anatomic  (not  mere  bacillary)  foothold,  is  ever  entirely 
wiped  out. 

It  is  unfortunate  that  in  the  above  series  I  have  micro- 
scopic sections  of  the  glands  of  only  one  animal;  namely,  of 
No.  28  (Ri),  killed  six  hundred  and  seventy-nine  days  after 
infection.  In  this  animal  the  right  and  left  inguinal  glands 
and  the  retroperitoneal  glands  were  all  enlarged,  —  much 
more    than    usual    so    long    after    infection.      The    bronchial 


STUDIES   IN    IMMUNITY   TO   TUBERCULOSIS.  I J 

glands  were  not  enlarged  and  their  consistence  and  appear- 
ance were  normal.  There  was  no  visceral  disease.  The 
inguinal  glands  were  hard  and  sclerotic,  but  showed  no  case- 
ating  points  to  the  naked  eye.  A  histological  examination 
of  these  glands,  however,  revealed  minute  tubercles  (giant 
cells  and  fine  patches  of  necrosis  set  in  the  center  of  dense 
masses  of  fibrous  tissue). 

Now  I  have  often  examined  the  glands  of  other  Ri  guinea- 
pigs  long  after  infection.  Many  were  normal  to  the  naked 
eye.  Some  which  appeared  almost  normal  showed  no 
anatomical  tubercle,  yet  revealed  a  cross  section  made  up 
wholly  of  scar  tissue.  Only  recently  I  examined  micro- 
scopically sections  of  glands  of  guinea-pigs  that  had  been 
infected  with  Ri  six  hundred  and  ninety-seven  days  previ- 
ously. They  showed  no  anatomical  tubercle.  There  was 
nothing  in  fact  except  dense,  hyaline,  scar  tissue.  But  the 
possibility  always  exists  that  somewhere  tubercle,  however 
slight,  may  be  lurking,  sufficient  to  set  up  at  least  a  mild 
skin  reaction.  It  is  a  remarkable  phenomenon,  and  one  that 
has  often  been  noted  in  the  human  body,  that  although  the 
body  can  keep  tubercle  under  perfect  control,  it  cannot 
eradicate  it  entirely. 

I  have  a  very  small  number  of  observations  in  which  I 
studied  the  effects  of  re-infection  on  a  decreasing  power  to 
react  cutaneously. 

14.  The  effects  of  re-infection  on  cutaneous  hypersensi- 
tiveness.  —  Numbers  28,  30,  and  31  reacted  six  hundred  and 
sixty-nine  days  after  infection,  as  described  in  Section  13. 
At  that  time,  April  20,  191 5 ,  they  were  given  inoculations  of 
virulent  H37  bacilli  in  the  skin.  Ten  days  later,  April  30, 
Nos.  30  and  31  were  again  tested  intracutaneously  with  the 
following  results : 

Twenty-four  hours :  No.  30,  Redness  and  induration,  30x30;  Indu- 
ration, -| — I — K  No.  31,  Redness  and  induration,  25  x  25  ;  Induration, 
+  +  +• 

48  hours  :  No.  30,  Redness  and  induration,  20  x  20 ;  Induration,  H — \-. 
No.  31,  Redness  and  induration,  15x15;   Induration,  -\ — ( — (-. 


1 8  KRAUSE. 

It  will  be  noted  that  re-infection  definitely  increases  the 
capacity  to  react  once  this  is  diminishing.  I  have  also 
noticed  this  feature  in  studies  made  on  animals  that  were 
inoculated  with  living  non-pathogenic  acid-fast  bacilli.  Such 
bacilli  are  non-pathogenic,  inasmuch  as  they  do  not  cause 
progressive  disease  in  animals.  But  they  will  give  rise  to 
abscesses  at  the  point  of  inoculation  and  to  enlargements  of 
the  regional  glands.  The  results  of  inoculation  are  apparent 
much  earlier  than  when  tubercle  bacilli  are  inoculated. 
Tumor  (going  on  to  abscess)  appears  at  the  site  as  early  as 
two,  three,  or  four  days  after  inoculation.  But  the  effects  are 
much  more  transitory  than  when  real  tubercle  exists. 

On  June  17  I  inoculated  four  white  guinea-pigs  subcu- 
taneously  in  the  right  groin  with  one  cubic  centimeter  of  a  very 
heavy  emulsion  of  living  acid-fast  Timothy  bacilli.  On  June 
24,  seven  days  later,  there  were  large  swellings  in  the  groins 
of  all,  and  on  this  day  I  injected  each  animal  intracutaneously 
with  .2  cubic  centimeter  W.E.  179  (1  cubic  centimeter  =  .01 
gram).     The  results  were  as  follows: 

June  25  (24  hours)  :  No.  1,  Redness  and  induration,  20  x  20.  No.  2, 
Redness  and  induration,  25  x  20.  No.  3,  Redness  and  induration, 
20  x  15.     No.  4,  Redness  and  induration,  25  x  20. 

June  26  (48  hours)  :  No.  1,  Redness  and  induration,  12  x  12.  No.  2, 
Redness  and  induration,  6x6.  No.  3,  Redness  and  induration,  6x6. 
No.  4,  Redness  and  induration,  10  x  10. 

On  July  8,  twenty-two  days  after  inoculation,  there  were 
no  palpable  signs  of  tumor  in  the  right  groin,  except  that  the 
glands  were  slightly  enlarged.  On  this  day  I  again  injected 
each  animal  intracutaneously  with  .2  cubic  centimeter  W.E. 
179  ( 1  cubic  centimeter  =  .01  gram)  and  obtained  the  fol- 
lowing results : 

July  9  (24  hours)  :  No.  1,  Pallid  type  of  reaction,  20  x  20  ;  Induration 
slight.  No.  2,  Negative.  No.  3,  Pallid  type,  30  x  30  ;  Slight  induration. 
No.  4,  Pallid  type,  35  x  20 ;  Slight  induration. 

July  10  (48  hours)  :     Nos.  1,  2,  3,  4,  All  reactions  completely  faded. 

In  this  experiment  the  noteworthy  points  are:  1,  the 
early  appearance  of  cutaneous  hypersensitiveness  (coincident 


STUDIES    IN    IMMUNITY   TO   TUBERCULOSIS.  1 9 

with  early  manifestation  of  lesions)  ;  and,  2,  the  transitory- 
character  of  the  hypersensitiveness. 

On  July  ill  again  inoculated  Nos.  I  to  4  subcutaneously 
in  the  left  groin,  each  with  1  cubic  centimeter  of  a  heavy 
emulsion  of  Timothy  bacillus. 

Three  days  later,  on  July  14,  all  received  intracutaneous 
injections  of  .2  cubic  centimeter  W.E.  1 79  ( 1  cubic  centi- 
meter =  .01  gram)  with  the  following  results: 

July  15  (24  hours)  :  All  animals  show  good  skin  reactions.  Indura- 
tion and  redness  are  marked.  In  all  guinea-pigs  they  measure  25  x  25. 
These  reactions  are  the  best  obtained  thus  far. 

From  these  experiments  it  would  appear  that  re-infection 
tends  to  increase  the  capacity  of  the  skin  to  react. 

15.  The  influence  of  the  general  tuberculin  reaction  on 
cutaneous  hypersensitiveness.  —  The  cutaneous  reaction,  the 
focal  reaction  (and  consequent  general  tuberculin  reaction), 
and  the  anaphylactic  reaction  to  tuberculo-protein  are  gen- 
erally looked  upon  as  being  different  phases  of  hypersensi- 
tiveness to  tuberculo-protein,  but  whether  they  are  depend- 
ent on  the  same  fundamental  causes  is  not  understood.  In 
the  following  experiments  the  effect  of  the  general  tubercu- 
lin reaction  on  cutaneous  hypersensitiveness  has  been  studied. 

In  Section  6  was  given  the  record  of  the  skin  reactions 
obtained  on  H37  guinea-pigs  on  August  5,  1913,  thirty-eight 
days  after  infection.  On  August  7  the  animals,  Nos.  40  to 
43,  were  injected  intraperitoneally,  each  with  one  cubic  centi- 
meter W.E.  [79.  The  next  day,  August  8,  they  were  notice- 
ably ill  and  were  then  injected  intracutaneously  with  .2  cubic 
centimeter  W.E.  179  (1  cubic  centimeter  =  .01  gram). 

The  following  results  were  noted  : 

August  9  (24  hours)  :  No.  40,  Pallid  reaction,  15x15;  Induration, 
slight.  No.  41,  Pallid  reaction,  15  x  r5  ;  Induration,  very  slight.  No. 
42,  Negative.     No.  43,  Pink  area,  12  x  12;  No  induration. 

August  10  (48  hours)  :  No.  40,  Negative.  No.  41,  Slight  redness  and 
induration.     No.  42,  Negative.     No   43,  Negative. 


20  KRAUSE. 

In  Section  7  are  detailed  the  effects  of  intracutaneous  tests 
on  Nos.  44  to  62,  made  on  August  13,  forty-six  days  after 
infection.  On  August  15  these  guinea-pigs,  Nos.  44  to  62, 
each  received  I  cubic  centimeter  W.E.  179  intraperitoneally. 
During  the  night  Nos.  50,  5  I  and  53  died  and  several  became 
very  ill.  The  next  day,  August  16,  several  of  the  stronger 
were  selected  for  intracutaneous  test,  and  reacted  as  follows : 

August  16  (24  hours):  No.  44,  Redness  and  induration,  25x25; 
Induration,  -f-  -\ — f- ;  Necrosis,  5x5.  No.  46,  Redness  and  induration, 
20x20;  Induration,  H — K  No.  49,  Redness  and  induration,  30x30; 
Induration,  -\ — | — (- ;  Necrosis,  10  x  10.  No.  54,  No  reaction;  Very 
weak. 

August  17  (48  hours)  :  No.  44,  Faded.  No.  46,  Faded.  No.  49, 
Faded.     No.  54,  Dying;  Killed. 

It  would  seem  that  there  is  no  doubt  that  the  general  reac- 
tion blunts  skin  hypersensitiveness  considerably.  We  usually 
conceive  of  the  general  reaction  as  being  due  to  the  absorp- 
tion of  focal  products  (focal  reaction).  The  result  here  is 
comparable  to  what  we  often  find  during  the  height  of  a 
recrudescence  of  tuberculous  disease.  Then,  during  the 
febrile  period,  when  more  or  less  absorption  from  the  focus 
is  taking  place,  the  cutaneous  hypersensitiveness  is  frequently 
in  complete  abeyance. 

General  discussion.  —  The  reader  who  is  familiar  with  the 
general  principles  of  immunity  to  tuberculous  re-infection 
must  have  already  noted  the  many  points  at  which  the  fac- 
tors underlying  cutaneous  hypersensitiveness  approach  those 
of  immunity  to  re-infection.  Some  of  these  general  princi- 
ples of  immunity  may  be  summarized  as  follows: 

1 .  There  is  no  immunity  without  a  preexisting  tuberculous 

focus. 

2.  Immunity  begins  to   manifest  itself  with  the  develop- 

ment of  the  focus. 

3.  It  decreases  with  the  healing  of  the  focus. 

4.  It  disappears  with  the  enucleation  of  the  focus. 

5.  The  degree  of  immunization  varies  directly  with  the 

virulence  of  the  immunizing  microorganism. 


STUDIES    IN    IMMUNITY   TO   TUBERCULOSIS.  2  1 

From  the  above  work,  as  well  as  from  the  researches  of 
Baldwin6  and  Romer,7  it  would  appear  that : 

1.  There    is   no    cutaneous   hypersensitiveness  without  a 

focus  (tubercle). 

2.  This  hypersensitiveness   appears   coincident  with   the 

establishment  of  the  focus. 

3.  It  diminishes  with  the  healing  of  the  focus. 

4.  It  varies    directly   with   the   intensity   of  the  disease, 

which  in  its  turn  is  dependent  on  the  virulence  of 
the  invading  bacillus. 

Whether  immunity  to  re-infection  and  cutaneous  hyper- 
sensitiveness are  both  merely  expressions  of  focal  activity, 
having  no  interrelationship,  or  whether  there  is  a  causal  rela- 
tionship between  the  two  phenomena  are  problems  that 
remain  to  be  investigated.  At  the  present  time  we  are 
engaged  on  work  the  aim  of  which  is  to  discover  whether 
immunity  to  re-infection  is  a  function  of  hypersensitiveness. 
The  expression  of  cutaneous  hypersensitiveness,  as  it  mani- 
fests itself  to  the  observer,  is  the  ability  of  the  skin  of  the 
affected  animal  to  react  in  an  inflammatory  manner  —  a  capa- 
city which  the  non-infected  organism  does  not  possess ;  and 
our  immediate  point  of  attack  is  the  meaning  and  nature  of 
this  inflammatory  reaction. 

Of  more  immediate  importance  is  why  the  numerous  cli- 
nicians who  have  investigated  the  cutaneous  reaction  on  man 
have  arrived  at  such  hopelessly  contradictory  results  as 
regards  the  interpretation  of  the  reaction.  The  answer  does 
not  seem  particularly  difficult.  We  must  remember  that  as 
pulmonary  tuberculosis  presents  itself  for  clinical  observation 
it  comes  to  the  clinician  as  a  disease  really  grounded  on  a 
very  complex  anatomical  basis.  We  may  take  for  granted 
that  at  bottom  the  simplest  case  is  a  mixture  of  very  varied 
anatomical  and  physiological  conditions;  a  jumble  of  foci  in 
all  stages  of  proliferative,  exudative,  and  degenerative  pro- 
cesses, continually  changing  one  into  the  other,  and  thereby 
altering  the  amount  and  the  time  element  of  give  and  take 
between  foci  and  host.     Again,  unless  violent  alterations  are 


22  KRAUSE. 

taking  place  in  the  diseased  areas,  the  net  changes  take 
unconscionably  long  periods  to  express  themselves  and  to 
make  themselves  apparent  to  the  observer.  It  is  also  evident 
that  it  is  very  difficult  to  measure  an  inflammation  or  the 
effect  by  which  cutaneous  hypersensitiveness  manifests  itself. 
It  is  not  easy  to  tabulate  differences  of  induration  and  differ- 
ent degrees  of  hyperemia.  It  is  only  comparatively  gross 
changes  that  are  appreciable  by  us.  To  compare  the  results 
in  one  patient  with  those  in  another  brings  us  no  useful 
knowledge.  At  any  particular  time  the  same  intensity  of 
reaction  in  two  or  three  different  patients  might  mean  two  or 
three  entirely  different  things.  But  some  information  might 
be  gained  by  a  prolonged  and  intensive  study  of  the  fluctu- 
ations of  hypersensitiveness  of  each  individual  patient,  by 
correlating  observations  of  the  reaction  with  those  obtained 
by  every  diagnostic  adjuvant.  Holmes8  has  attempted  some- 
thing of  this  kind.  He  has  studied  individuals  and  has  com- 
pared their  reactions,  one  with  another;  and  he  has  arrived 
at  results  that  resemble  what  we  have  obtained  from  animal 
experimentation.  He  concludes  that  "  individuals  with  pul- 
monary tuberculosis  present  marked  tuberculin  hypersensi- 
tiveness (cutaneous)  ;  this  diminishes  if  they  improve,  fluctu- 
ates, or  remains  stationary,  if  they  do  not,  rises  if  they  fail." 
It  has  also  seemed  to  me  that  Hamman  9  displayed  unusual 
acumen  when  he  wrote :  "  We  believe  that  tuberculin  hyper- 
sensitiveness in  relation  to  tuberculous  disease  runs,  roughly, 
somewhat  as  follows :  Since  nearly  all  adults  are  infected 
with  tuberculosis,  we  assume  a  low  grade  of  tuberculin- 
hypersensitiveness  to  begin  with.  Should  there  be  a  fresh 
invasion  of  the  body  from  within  or  from  without,  the  tuber- 
culin-hypersensitiveness  rapidly  rises.  If  the  disease  sub- 
sides and  the  individual  recovers  the  hypersensitiveness 
gradually  falls  to  a  lower  level,  perhaps  to  the  original  low 
level;  if  the  disease  remains  active,  the  high  level  of  hyper- 
sensitiveness persists  and  lasts  until  the  body  is  overwhelmed 
and  its  resistance  broken  down  completely  by  the  disease, 
when  hypersensitiveness  disappears." 


STUDIES    IN    IMMUNITY    TO    TUBERCULOSIS.  23 

SUMMARY. 

1.  Cutaneous  hypersensitiveness  to  tuberculo-protein  is 
inaugurated  by  the  establishment  of  infection  and  the 
development  of   the   initial  focus. 

2.  It  increases  with  progressive  disease. 

3.  It  varies  directly  with  the  extent  and  intensity  of  the 
disease. 

4.  It  diminishes  with  the  healing  of  the  disease. 

5.  It  is  probably  never  entirely  lost  (except  in  the 
presence  of   intercurrent  disease,  pregnancy,  etc.). 

6.  It  is  increased  by  re-infection. 

7.  It  is  diminished  or  completely  wiped  out  during  the 
period  of   the  general  tuberculin   reaction. 

8.  It  is  suggested  that  tissue  hypersensitiveness  may  be  a 
function  of  immunity  to  re-infection. 

[This  work  was  made  possible  through  the  generosity  of  Mrs.  A.  A. 
Anderson  of  New  York  City.] 

BIBLIOGRAPHY. 

1.  Mendel.     Med.  Klinik,  1908,  12. 

2.  Mantoux  and  Moussu.     Acad,  des  Sc,  Sept.  14,  1908. 
Mantoux.     La  Presse  Med.,  1910,  No.  2,  10. 

3.  Romer.     Beitr.  z.  Klin.  f.  Tuberc,  1909,  xiv,  1. 

4.  Baldwin.  Trans.  Nat.  Assoc.  Study  and  Prev.  of  Tuberc,  7th  Ann. 
Meeting,  191 1,  352. 

5.  Romer.     Beitr.  z    Klin.  f.  Tuberc,  1910,  xvii,  287. 

6.  Baldwin.     Loc  cit. 

7.  Romer.     Beitr.  z.  Klin.  f.  Tuberc,  1909,  xiv,  1. 

8.  Holmes.  Trans.  Nat.  Assoc.  Study  and  Prev.  of  Tuberc,  10th  Ann. 
Meeting,  1914,  133. 

9.  Gelien  and  Hamman.  Bull.  Johns  Hopkins  Hosp.,  1913,  xxiv, 
No.  268,   180. 


Studies  in  Immunity  to  Tuberculosis.* 

the  anaphylactic  state  in  its   relation  to   resist- 
ance to  tuberculous  infection  and  tuberculous 

DISEASE. 

AN    EXPERIMENTAL   STUDY. 

A.  K.  Krause,  M.D. 
{From  the  Saranac  Laboratory  for  the  Study  of  Tuberculosis.) 

The  one  outstanding  development  of  our  hard-won  knowl- 
edge of  tuberculosis  during  the  last  ten  years  is  the  rapidly 
growing  belief  that  tuberculous  infection  and  the  disease 
tuberculosis  are  two  entirely  different  phases  of  an  inter- 
dependent situation.  It  required  the  widespread  application 
of  the  cutaneous  tests  and  the  further  discovery  of  the  fact 
that  the  positive  cutaneous  reactions  occurred  only  in  the 
presence  of  tubercle,  to  clinch  the  pathologists'  contention 
that  a  large  proportion  of  civilized  mankind  is  tainted  with 
tubercle,  yet  does  not  suffer  from  tuberculosis.  (Throughout 
this  paper  the  term  "  tubercle  "  is  used  to  denote  merely 
anatomic  change,  while  the  term  "  tuberculosis  "  is  used  to 
denote  clinical  disease.)  It  followed  further,  as  a  natural 
and  logical  conclusion,  that  tuberculous  infection  or  the 
initial  development  of  tubercle  does  not  carry  with  it  the 
necessity  of  the  immediate  development  of  clinical  tubercu- 
losis. The  great  majority  of  people  who  have  tubercle  pass 
through  a  long  life  reacting  positively  to  infection  tests;  yet 
with  bacilli  in  some  way  or  other  under  control,  they  never 
show  symptoms  of  specific  illness.  In  the  same  way,  but  to 
a  lesser  degree,  they  are  in  the  same  condition  as  the  man 
who  once  had  clinical  tuberculosis,  whose  disease  has  become 
arrested  and  who  lives  through  a  shorter  or  longer  interval  — 
perhaps  through  the  rest  of  a  long  life  —  without  ever  again 
experiencing  a  symptom  of  tuberculosis.  We  know  that 
such  a  man  harbors  hidden  sources  of  danger  to   himself,  we 

*  Received  for  publication  June  10,  1916. 
(25) 


26  KRAUSE. 

know  too  that  he  will  always  react  to  tuberculin  tests.  We 
cannot  guarantee  that  the  old,  once  active  foci  will  never 
again  flare  up.  We  can  only  tell  him  that  so  long  as  his 
foci  of  disease  and  the  bacilli  which  they  often  undoubtedly 
contain  remain  well  walled  off  and  encysted,  just  so  long  he 
is  likely  to  be  free  from  tuberculosis. 

It  follows,  too,  that  the  tuberculosis  of  adolescents  and 
adults  in  many  cases  develops  from  benign  tubercle  that  has 
been  implanted  earlier  in  life.  In  other  words,  tuberculosis 
often  originates  from  a  preexistent  benign  focus.  Although 
there  are  a  great  many  facts  to  support  this  view,  I  shall 
content  myself  here  with  merely  making  the  assertion  as  a 
matter  of  fact.  I  would  remind  the  reader  that  bone  and 
joint,  adrenal,  and  meningeal  tuberculosis  must  develop  from 
some  primary  focus,  whether  this  primary  focus  is  demon- 
strable or  not.  And  I  would  further  remind  him  that  such 
tuberculosis  frequently  develops  in  those  who  have  hitherto 
enjoyed  perfect  health. 

If  now  we  grant  that  tubercle  is  very  commonly  implanted 
in  early  life,  that  tuberculosis  is  usually  not  an  immediate 
consequence,  that  the  majority  of  people  go  through  life 
with  tubercle,  but  without  tuberculosis,  and  that  in  a  great 
many  cases  tuberculosis  develops  from  preexisting  tubercle, 
it  becomes  of  the  utmost  importance  that  we  acquire  some 
well-controlled  information  concerning  the  conversion  of 
tubercle  into  tuberculosis.  In  the  light  of  our  newer  knowl- 
edge, too,  the  conditions  that  influence  infection  and  the 
further  dissemination  of  tubercle  need  much  more  study 
than  they  have  thus  far  received. 

Once  tuberculous  infection  is  established  in  an  animal  it  is 
obvious  that  numerous  mechanical  factors,  alone  or  working 
with  other  forces,  will  determine  whether  the  initial  focus 
will  remain  localized  or  whether  dissemination  from  it  will 
take  place.  As  example  of  such  influences  I  need  only 
mention  the  site  of  the  initial  focus,  its  degree  of  fibrous 
investment  or  the  character  of  its  center,  whether  more  or 
less  caseous,  more  or  less  fibrous  or  calcareous,  variations  in 
the  blood  and  lymph  flow  to  and  from  the  focus  as  these  are 


STUDIES   IN    IMMUNITY   TO   TUBERCULOSIS.  27 

determined  by  a  multiplicity  of  physical  conditions.  If  for 
the  time  being  we  leave  out  of  consideration  these  well- 
recognized  mechanical  factors,  we  then  assume  that  infection 
and  the  further  extension  of  tubercle  are  also  modified  by 
qualities  that  appertain  to  one  or  both  of  the  two  organisms 
concerned  ;  namely,  the  invader  and  the  host,  the  tubercle 
bacillus  and  the  infected  individual. 

It  is  generally  held  that  the  issue  of  tuberculous  infection 
turns  on  the  one  hand  upon  the  number  and  virulence  of  the 
bacilli  and  the  poisons  elaborated  by  them  or  inherent  in 
them,  and,  on  the  other  hand,  upon  the  susceptibility  or 
resistance  of  the  body.  Some  at  least  of  these  attributes 
need  elucidation.  It  is  becoming  more  and  more  generally 
accepted  to-day  that  the  tubercle  bacillus  contains  or  elabo- 
rates no  specific  poison  or  toxin,  but  that  the  various  symp- 
toms of  intoxication  met  with  in  tuberculosis  are  due  to 
bacillary  protein  sensitization  and  intoxication  in  their 
various  forms.  Therefore,  what  was  formerly  looked  upon 
as  being  due  purely  to  the  effects  of  a  bacillary  attribute,  is 
now  viewed  as  being  dependent  in  part  on  the  changed 
reactions  of  the  body  which  help  to  make  up  that  complex 
and  elusive  condition  that  we  are  pleased  to  call  resistance. 

There  is  no  doubt  that  ceteris  paribus,  the  number  of 
bacilli  concerned  at  any  time  does  have  a  marked  influence 
on  the  degree  and  extent  of  infection.  This  fact  can  be 
easily  established  by  simple  experiment  and  can  be  dismissed 
in  a  word.  But  whether,  as  is  often  assumed,  the  virulence 
of  tubercle  bacilli,  as  they  occur  in  disease,  is  so  very 
variable,  is  not  altogether  so  clear.  There  is  no  doubt  that 
strains  that  have  been  isolated  from  the  animal  body  fre- 
quently decrease  in  virulence  once  they  are  trained  to  a 
vegetative  test-tube  existence.  This  every  observer  of  any 
experience  must  have  noted.  But  it  has  been  the  experience 
of  the  Saranac  Laboratory  that  this  lost  virulence  is  never 
regained,  though  a  variety  of  well-known  methods  have  been 
tried  in  the  effort  to  bring  back  original  virulence  to  a 
culture. 


28  KRAUSE. 

Variation  in  bacillary  virulence  has  often  been  brought  in 
to  explain  the  multiplicity  of  clinical  and  anatomical  types 
of  tuberculosis.  But  to  attempt  in  this  way  to  explain  too 
much  of  the  variety  of  clinical  finding  in  tuberculosis  would 
soon  involve  one  in  endless  contradiction.  As  a  matter  of 
fact,  most  of  the  studies  that  have  aimed  to  differentiate 
virulence  in  bacillary  strains  as  they  have  been  isolated  from 
tuberculous  disease  of  many  varieties  and  in  many  locations 
have  not  been  very  fruitful  (Vagedes,1  Krompecher  and 
Zimmermann,2  C.  Frankel  and  Baumann,3  Moller,4  Burnet,5 
English  Commission,  etc.).  They  have  at  least  not  brought 
to  light  the  variations  in  virulence  that  many  writers  have 
too  often  assumed.  Perhaps  our  technical  methods  have 
heretofore  been  too  coarse.  At  any  rate,  satisfactory 
information  on  this  point  is  much  to  be  desired  and  it 
should  be  the  subject  of  prolonged  and  carefully  controlled 
research,  for  its  importance  can  hardly  be  exaggerated. 

Although  a  great  deal  of  laborious  work  has  failed  to 
bring  forward  much  definite  knowledge  of  the  variations  in 
virulence,  as  these  arise  in  tuberculous  man  and  animals,  the 
most  casual  observer  has  noted  that  once  infection  is  estab- 
lished its  subsequent  course  is  largely  dependent  on  fluctua- 
tions in  the  resistance  or  susceptibility  of  the  individual  —  in 
other  words,  in  the  way  in  which  at  any  given  time  the  indi- 
vidual reacts  to  the  invader.  We  see  the  individual  reacting 
now  exudatively,  now  proliferatively  to  his  infection.  By 
the  application  of  the  cutaneous  test  we  can  discover  very 
prettily  that  the  reaction  of  at  least  one  of  his  tissues,  the 
skin,  can  be  a  very  changeable  one.  What  underlies  these 
changes  we  do  not  know  :  the  fact  remains  that  they  are 
there. 

It  is  very  probable  that  man  has  very  little  native  resist- 
ance to  tuberculous  infection.  Evidence  is  continually  accu- 
mulating that  if  exposed  to  the  living  tubercle  bacillus,  every 
man  will  become  infected.  How  else  explain  our  autopsy 
and  cutaneous  test  results  which  show  tubercle  to  be  such  a 
universal  taint?  But  there  is  a  very  real  and  a  very  impor- 
tant difference  in    the  way  in  which    individuals  react  to  this 


STUDIES    IN    IMMUNITY   TO    TUBERCULOSIS.  29 

infection.  This  observation  is  too  patent  to  need  demonstra- 
tion. This  is  the  difference  between  what  we  call  resistance 
on  the  one  hand,  and,  on  the  other,  lack  of  resistance.  The 
term  resistance  and  its  opposite  are  vague  terms,  yet  their 
manifestations  are  none  the  less  real.  We  know  that  we  can 
diminish  or  increase  resistance  by  certain  hygienic  measures: 
one  of  Dr.  Trudeau's6  first  experimental  works  showed  this. 
Murphy's7  recent  work  on  the  effects  of  radiation,  and 
Arloing's,s  and  Lewis  and  Margot's9  on  those  of  splenectomy, 
also  illustrate  the  point.  The  conditions  of  the  experiments 
of  Lewis  and  Murphy  are,  however,  such  as  can  hardly  occur 
in  natural  infection  and  are  only  valuable  in  so  far  as  they 
give  us  an  insight  into  the  fundamental  causes  of  immunity 
in  tuberculosis. 

In  the  experiments  on  resistance  and  virulence  which  I 
shall  detail  I  have  had  in  mind  several  of  the  features  to 
which  I  have  referred.  For  the  purposes  of  infection  I  have 
used  several  strains  of  tubercle  bacilli  with  which  I  have 
worked  constantly  for  seven  years,  and  whose  constancy  of 
virulence  under  various  circumstances  I  am  familiar  with  and 
fairly  certain  of.  With  one  of  them,  Strain  Ri,  I  aimed  to 
produce  a  non-progressive  and  self-healing  tuberculosis  in 
guinea-pigs,  such  as  we  see  approximated  in  the  non-pro- 
gressive or  quiescent  infection  of  man.  I  then  sought  for  a 
medium  which  might  presumably  reduce  the  resistance  of 
my  animals  to  infection  and  further  invasion,  a  medium  which 
I  desired  to  be  comparable  to  what  we  may  meet  with  in 
man  under  natural  conditions.  By  the  application  of  this 
medium  I  tried  to  convert  a  non-progressive  or  self-limited 
tuberculosis  into  a  progressive  and  spreading  infection.  My 
endeavor  was  to  study  some  small  possible  element  in  resist- 
ance, some  element  that  was  sharp  and  definite  and  easily 
controlled. 

The  medium  which  I  selected  was  non-lethal  anaphylactic 
shock.  It  is  a  common  clinical  experience  to  find  tubercu- 
losis developing  soon  after  some  other  acute  infection,  such 
as  measles,  tonsilitis  or  catarrhal  affections  of  the  respiratory 
tract.     It   is  conceivable,   and   indeed    probable,  that  during 


30  KRAUSE. 

the  course  of  the  measles  or  the  influenza,  local  circulatory 
conditions  (congestion,  etc.)  are  set  up  around  an  already 
existing  focus  of  tuberculous  infection,  that  favor  the  mobil- 
ization and  dissemination  of  bacilli  from  what  was  hitherto  a 
benign  tubercle.  But  that  this  is  not  the  whole  story  is  sug- 
gested by  the  fact  that  during  an  attack  of  measles  or  during 
the  febrile  period  of  other  infections,  a  patient  who  formerly 
reacted  positively  to  the  cutaneous  tuberculin  tests  often 
loses  his  skin  sensitiveness.  This  loss  of  skin  sensitiveness  is 
also  not  infrequently  met  with  during  an  acute  febrile  exacer- 
bation of  tuberculosis  in  the  tuberculous  patient  himself. 
We  must  assume,  therefore,  that  during  intercurrent  infection 
or  renewed  activity  of  tuberculosis,  the  specific  immunity 
reaction  to  tuberculin  that  is  represented  by  cutaneous 
hypersensitiveness  may  be  in  abeyance. 

Now  both  the  cutaneous  tuberculin  reaction  and  the  gen- 
eral anaphylactic  reaction  are  phases  of  hypersensitiveness 
to  protein.  Furthermore,  the  idea  developed  by  Vaughan 
that  many  of  the  constitutional  symptoms  of  infection,  of 
which  fever  is  such  a  prominent  one,  are  due  to  intoxication 
by  dissociated  proteins,  which  in  turn  depends  on  the  hyper- 
sensitiveness of  an  organism  that  has  become  allergic  to  the 
specific  protein  under  consideration,  is  gaining  more  and 
more  adherents.  It  was  therefore  but  a  step  to  reason  that 
the  resistance  of  the  body  to  tuberculous  infection  and  exten- 
sion might  be  reduced  by  protein  intoxication  or  anaphy- 
laxis. 

Several  years  ago  I  10  had  done  a  few  experiments  that  led 
me  to  believe  that  such  might  be  the  case.  At  that  time  I 
studied  the  relation  between  hypersensitiveness  to  tuberculo- 
protein  and  immunity  to  tuberculous  infection.  If  my  non- 
tuberculous  animals  were  first  artificially  sensitized  to 
tuberculo-protein  and  then  inoculated  with  living  tubercle 
bacilli,  after  a  proper  length  of  time  I  could  not  determine 
that  sensitized  animals  reacted  to  infection  any  differently 
from  non-sensitized  normal  controls.  If,  however,  a  few  of 
the  sensitized  were  given  a  non-lethal  toxic  injection  of 
tuberculo-protein,  and  if  they  developed  symptoms  of  acute 


STUDIES    IN    IMMUNITY    TO    TUBERCULOSIS.  3  I 

anaphylaxis  and  were  then  very  shortly  afterwards  inoculated 
with  living  bacilli  it  struck  me  that  the  disease  extended 
further  than  was  the  case  in  either  the  merely  sensitized 
animals  or  in  the  normal  controls.  As  a  result  of  these 
experiments  I  published  the  following  conclusions:  (1) 
"  Sensitization  of  non-tuberculous  guinea-pigs  with  tuber- 
culo-protein  does  not  alter  their  resistance  to  experimental 
tuberculous  infection;  "  and  (2)  "  Resistance  to  infection  is 
markedly  lowered  during  the  period  that  a  sensitized  animal 
is  suffering  from  symptoms  of  anaphylactic  shock." 

In  these  experiments  of  191 1  the  microorganism  used  for 
infection  was  our  strain,  H37  tubercle  bacillus,  which  is  a 
virulent  human  bacillus  and  produces  progressive  tubercu- 
losis in  guinea-pigs.  I  could  interpret  my  results  only  by 
comparing  the  extent  of  tuberculous  involvement  in  the 
different  sets  of  animals  at  a  given  time  after  inoculation. 
In  any  experiments  of  this  kind,  unless  the  differences  were 
sharp  and  constant  and  the  number  of  animals  was  large, 
any  interpretation  would  always  be  open  to  justifiable  criti- 
cism. As  a  matter  of  fact,  the  differences  in  these  particular 
experiments  were  fairly  marked  and  uniform,  but  the  number 
of  animals  used  never  satisfied  me  as  being  sufficient  to 
overcome  all  sources  of  error  in  interpretation.  I  therefore 
determined  to  repeat  the  experiment  under  conditions  that 
could  be  more  easily  controlled  and  more  sharply  differenti- 
ated. 

The  microorganism  used  for  infection  in  the  following 
experiments  was  our  strain,  Ri  human  bacillus.  This  germ 
was  isolated  by  Dr.  Trudeau  in  1891.  After  a  few  years  of 
incubator  existence  (about  1893)  its  virulence  for  guinea- 
pigs  dropped  to  a  level  where  it  has  remained  constant  ever 
since.  Subcutaneous  inoculation  into  the  groin  of  a  guinea- 
pig  will  bring  about  hyperplasia  and  caseating  tuberculosis 
of  the  regional  glands,  as  well  as  of  the  iliac  glands,  but 
macroscopic  tuberculosis  of  any  of  the  abdominal  and 
thoracic  viscera  occurs  infrequently.  Only  under  excep- 
tional conditions,  pregnancy,  intercurrent  disease,  excessive 
or  repeated  dosage,  will  macroscopic  tubercle  develop   in  the 


32  KRAUSE. 

spleen,  or  with  extreme  rarity  in  the  lungs  or  liver.  Six 
months  or  a  year  after  inoculation  the  superficial  inguinal 
glands,  which  were  once  enlarged,  begin  to  return  to  their 
normal  size  and  gradually  approach  their  normal  consistence. 
I  have  carried  some  guinea-pigs  that  I  inoculated  subcu- 
taneously  with  Ri  for  two  years  and  more.  At  the  end  of 
this  time  I  found  all  viscera  clean  and  normal  to  the  naked 
eye,  the  iliac,  and  deep  inguinal  glands  apparently  normal, 
and  with  the  exception  of  slight  enlargement  and  radi- 
ating, translucent  grayish  lines  and  here  and  there  an  opaque 
yellowish-white  point,  the  superficial  inguinal  glands  were 
almost  normal  in  appearance.  Cutaneous  tests  before  death, 
however,  proved  such  animals  to  be  still  hypersensitive  to 
tuberculo-protein,  and  histological  examination  revealed 
microscopic  tuberculosis  in  the  superficial  inguinal  glands. 
One  and  two  years  after  inoculation  the  animals  are  always 
sleek,  fat,  lively,  and  in  apparent  perfect  health,  though  they 
harbor  a  localized  tuberculosis  and  react  to  tuberculo-protein. 
We  have,  therefore,  always  looked  upon  Ri  infected  animals 
as  affected  with  self-limited,  healing  tuberculosis,  and  more 
or  less  comparable  to  those  human  beings  who  are  infected, 
but  whose  infection  shows  no  tendency  to  progress.  In  the 
first  series  of  our  new  experiments  one  set  of  animals  was 
first  sensitized  with  tuberculo-protein,  then  anaphylactized, 
and  finally  infected  with  Ri.  Another  set  was  first  sensi- 
tized, "then  infected  with  Ri  and  finally  anaphylactized. 
Both  sets  were  then  allowed  to  live  for  a  certain  length  of 
time,  after  which  the  effects  of  anaphylactic  shock  on  infec- 
tion and  the  spread  of  the  disease  were  studied. 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  33 

Experiment  i. 

1 .  Sensitization  of  guinea-pigs  : 

May  12  and  13:  Nos.  1  to  17,  each  injected  intraperitoneally  with  2 
cubic  centimeters  W.E.*  (water-extract)  179. 

2.  Toxic  injection  of  animals  : 

June  17  and  18:  Nos.  1  to  17  injected  post-orbitally  with  E.E.* 
(extract-emulsion)  179,  in  doses  varying  from  .2  to  .5  cubic  centimeter. 

Results:  Nos.  5  and  15  died  of  anaphylactic  shock.  Nos.  1,  9,  and 
10,  no  symptoms.  Nos.  4  and  8,  symptoms,  +;  recovered.  Nos.  3,  7, 
12,  13,  14  and  17,  symptoms,  -\ — (- ;  recovered.  Nos.  2,  6,  11  and  16, 
symptoms,  +  -\ — (-  ;   recovered. 

3      Inoculation  of  animals  with  Ri : 

June  18:  Emulsion  used:  140  milligrams  of  a  glycerin-beef-serum 
culture  of  Ri,  planted  June  1  and  showing  good  growth,  were  ground  in  a 
mortar  with  a  few  drops  of  .85  per  cent  physiological  salt  solution.  Ten 
cubic  centimeters  of  physiological  salt  solution  were  then  added  to  the 
mush  and  the  emulsion  was  centrifugated  until  all  large  clumps  were 
thrown  down.  The  supernatant  fluid,  which  was  milky,  was  then  used  for 
inoculation. 

Animals  inoculated:  Set  A:  Nos.  1  to  4,  sensitized  May  12;  intoxi- 
cated, June  17,  twenty-four  hours  before  inoculation. 

Set  B:  Nos.  6  to  10,  12  to  14,  16  and  17,  sensitized  May  12  and  13; 
intoxicated,  June  18,  one  to  three  hours  before  inoculation. 

Set  C  :  Nos  41  to  48,  Originally  normal  non-sensitized,  but  on  June  18 
given  post-orbital  injection  of  .25  cubic  centimeter  E.E.  179,  to  control 
the  effects  of  the  post- orbital  injection  on  the  infection.  Injection  of  E.E. 
given  one  hour  before  inoculation. 

Set  D  :  Nos.  49  to  56,  Normal  controls ;  no  preliminary  preparation 
before  inoculation. 

Inoculation :  Each  of  the  above-named  animals  was  inoculated  with 
.25  cubic  centimeter  of  the  above  emulsion,  subcutaneously  in  the  right 
groin. 

4.     Results : 

On  August  8,  fifty-one  days  after  Rr  inoculation,  all  the  infected  guinea- 
pigs  were  killed  and  autopsied.  The  findings  at  section  were  not  very 
conclusive.  At  the  end  of  my  notes  for  the  day  I  have  a  summary  in 
which  I  say,  "Differences  between  the  various  sets  of  animals  are  too 

*  W.E.  179 :  A  water  extract  of  Bac.  Tub.  H37,  containing  bacillary  protein. 
Prepared  as  follows  :  122  gms.  dried  H37  (from  glycerin-broth  cultures),  pulverized 
for  one  week  in  a  mill ;  powder  extracted  with  Aq.  dest.,  1,830  cc,  at  50°  C.  for  48  hrs. ; 
let  stand  3  days  in  the  dark ;  filtered  through  Berkefeld  filter.  The  filtrate  is  the  W.E. 
179.     1  cc.  of  dried  residue  =  .02  gm. 

E.E.  179 :  Is  a  small  portion  of  the  above  extract  withdrawn  before  filtration  and 
allowed  to  settle  out  in  the  dark.     1  cc.  dried  =  .022  gm. 


34  KRAUSE. 

slight  to  conclude  that  nqn-lethal  anaphylactic  shock  affected  the  animals1 
resistance.  The  two  most  advanced  lesions  are  in  Nos.  13  and  14 
(anaphylactized  animals),  which  had  the  added  burden  of  pregnancy." 

However,  careful  analysis  of  the  autopsy  details  of  each  animal  as  they 
are  set  down  reveals  the  fact  that  the  gross  tuberculous  involvement  was 
more  advanced  in  more  anaphylactized  animals  than  in  those  of  Sets  C 
and  D.  There  were  no  anaphylactized  animals  which  showed  as  little 
involvement  as  the  best  of  the  controls  {i.e.,  Nos.  41  to  56),  seven  of 
which  had  no  visceral  disease  whatever.  In  five  of  the  anaphylactized 
animals  the  lungs  showed  miliary  foci.  This,  a  very  rare  event  after  an 
ordinary  Ri  infection,  occurred  in  only  one  control.  In  only  one 
anaphylactized  animal,  No.  17,  was  the  disease  localized  strictly  to  the 
glandular  (lymphatic)  system.  In  seven  of  the  controls  it  was  so 
confined. 

If,  in  all  but  a  few  of  the  anaphylactized  animals  the  infection  had 
escaped  from  the  glandular  system,  while  in  most  of  the  controls  it  had 
been  limited  to  the  lymphatics,  I  should  have  no  hesitancy  in  concluding 
that  if  original  infection  (and  perhaps,  too,  dissemination  from  an  already 
existing  focus)  takes  place  during  the  phase  of  anaphylactic  shock,  it 
occurs  at  a  time  when  resistance  to  infection  is  lowered.  In  view  of  the 
results  of  this  experiment  I  can  do  no  more  than  suggest  that  this  is 
probably  the  case. 

It  must  be  remembered  that  these  animals  were  killed  fifty-one  days 
after  infection.  We  have  found  by  experience  that  Rr  infection  reaches 
its  maximum  and  maintains  it  at  from  thirty  or  forty  days  to  three  or  four 
months.  From  this  time  on,  under  ordinary  conditions,  the  disease  tends 
to  retrogress.  Enlarged  and  hard  glands  tend  to  diminish  in  size  and 
regain  some  of  their  normal  elasticity,  and  the  occasional  nodule  that  is 
seen  in  the  spleen  tends  to  disappear,  so  that  after  a  year  or  two  the 
naked  eye  can  detect  little  except  enlarged  and  partially  sclerotic,  super- 
ficial inguinal  glands.  It  is  impossible  to  say  now  whether  these  various 
sets  of  animals  would  have  shown  more  striking  variations  later  in  the 
disease.     I  hope  to  study  this  matter  further. 

Experiment  2. 
{Plan  :    To  sensitize  guinea-pigs  with  tuberculo-protein  ;  after  sensitive- 
ness is  established  to  infect  them  with  Ri  ;  after  disease  is  established  to 
give  the  originally  sensitized  ones  non-lethal  anaphylaxis  and  study  the 
effects  of  anaphylaxis  on  the  disease  already  present.) 

1 .  Sensitization  of  guinea-pigs  : 

May  15  :  Nos.  31  to  40,  each  injected  intraperitoneal^  with  2  cubic 
centimeters  W  E.  179. 

2.  Inoculation  of  animals  with  Ri  : 

August  9:  Emulsion  used:  A  spadeful  of  Ri  from  glycerin-beef- 
serum  culture,  good  growth,  planted  July  10,  rubbed  up  in   mortar  with  a 


STUDIES    IN   IMMUNITY   TO   TUBERCULOSIS.  35 

few  drops  of  physiological   salt   solution.     Emulsified   in   salt   solution. 
Not  filtered.     Emulsion  opalescent. 

Animals  inoculated  :     Set  A  :     Nos.  31  to  40,  sensitized  May  15. 

Set  B :     Nos.  67  to  76,  non-sensitized  normal  controls. 

3.  Tests  of  sensitiveness  of  animals  at  time  of  inoculation  : 

August  11  :  Nos.  18  to  20,  from  same  original  series  as  Nos.  31  to  40, 
and  sensitized  on  May  13,  were  to-day  given  post-orbital  injections  of 
E.E.  179.     They  proved  to  be  very  sensitive. 

4 .  Toxic  injections  of  animals  : 

September  30:  Fifty-two  days  after  inoculation  and  138  days  after 
sensitization  :  Nos.  31  to  40  injected  post-orbitally  with  E.E.  179,  in  doses 
of  from  .2  to  .25  cubic  centimeter.  All  had  -\ — (-  or  +  +  +  symptoms 
of  anaphylactic  shock  except  No.  37,  which  had  no  symptoms.  All 
recovered. 

5 .  Results  : 

On  December  3,  sixty-four  days  after  anaphylactic  shock  and  116  days 
after  inoculation  with  Ri,  all  the  infected  guinea-pigs  were  killed  and 
autopsied. 

There  was  no  appreciable  difference  between  the  two  lots.  None  of 
the  animals  had  visceral  disease.  All  showed  practically  healed  processes, 
with  slightly  enlarged  and  firm  regional  glands  the  only  evidences  of 
infection. 

The  animals'  resistance  to  disease  was  therefore  apparently  not  lowered 
by  the  anaphylactic  shock  which  they  had  experienced  some  time  before. 
Other  phases  of  this  experiment  will  be  taken  up  in  the  general  discussion 
at  the  end  of  this  paper. 

Experiment  3. 
(Plan:  Exactly  the  same  as  that  of  Experiment  1,  except  that  for 
purposes  of  infection  Moeller's  Mist  Bacillus  was  used  instead  of  the  Ri 
human  tubercle  bacillus.  As  is  well  known  the  Mist  Bacillus  is  one  of  the 
so-called  saprophytic  or  non-pathogenic  acid-fast  bacilli.  It  possesses 
practically  no  parasitism  for  mammals.  If  inoculated  subcutaneously 
into  guinea-pigs,  even  in  enormous  doses,  it  produces  an  abscess  at  the 
site  of  inoculation,  but  does  not  cause  lesions  in  the  viscera.  The 
regional  glands  also  undergo  some  hyperplasia.  Speaking  broadly,  we 
may  say  that  the  local  effects  differ  from  those  brought  about  by  the 
tubercle  bacillus,  in  that  it  takes  relatively  enormous  doses  of  the  Mist 
Bacillus  to  produce  any  appreciable  change  (abscess),  in  that  the  changes 
are  evident  in  from  two  to  three  days,  and  in  the  fact  that  they  are  very 
transient  and  non-progressive  as  compared  to  what  is  found  in  real  tuber- 
culosis. As  in  R 1  infection  the  aim  of  this  experiment  was  to  discover 
whether  anaphylactic  shock  would  so  reduce  the  resistance  of  the  body  as 
to  allow  a  saprophytic  microorganism,  the  Mist  Bacillus,  to  "  take  hold."  ) 


36  KRAUSE. 

i .     Technic  of  the  experiment : 

The  steps  followed  were  exactly  the  same  as  those  outlined  in  Experi- 
ment I,  except  that  the  time  intervals  were  somewhat  different.  The 
animals  were  intoxicated  with  E.E.  179,  forty-three  days  after  sensitization 
with  W.E.  179.  Inoculation  with  enormous  doses  of  an  emulsion  of 
Bac.  Mist  was  made  immediately  after  intoxication  on  six  guinea-pigs 
that  survived  the  toxic  dose  and  on  controls. 

2 .     Results : 

August  12.  The  animals  were  killed  forty-seven  days  after  inoculation. 
The  different  sets  of  animals  revealed  no  differences  of  involvement. 
A  few  of  each  set  had  slightly  enlarged  regional  glands.  The  rest  were 
entirely  normal.  Careful  search  of  stained  smears  of  some  of  the 
enlarged  glands  revealed  no  acid-fast  bacilli.  Anaphylactic  intoxication 
shortly  before  inoculation  apparently  did  not  alter  the  animals1  response 
to  the  parasitism  of  the  Mist  Bacillus. 

Experiment  4. 

At  the  time  that  we  were  engaged  on  some  of  the  experi- 
ments described  above,  Thiele  and  Embleton11  published  a 
work  entitled,  "  The  Pathogenicity  and  Virulence  of  Bacteria." 
They  reported  that  they  had  been  able  to  change  non-patho- 
genic into  pathogenic  bacteria  by  a  process  which  in  its  gen- 
eral principles  (except  the  time  element)  resembled  the  one 
which  I  have  already  described.  Their  usual  procedure  was 
as  follows:  They  would  sensitize  normal  guinea-pigs  with 
large  doses  of  a  non-pathogenic  microorganism  such  as  the 
Timothy  bacillus  and  the  Smegma  bacillus.  From  five  to 
fifteen  days  later  they  would  then  inoculate  these  animals  with 
smaller  doses  of  the  living  organism  of  the  same  type  and 
find  that  this  second  inoculation  caused  disease  and  death. 
They  reported  further  that  the  virulence  of  the  originally 
non-pathogenic  organism  was  so  enhanced  that  bits  of  tissue 
from  the  animals  thus  infected  produced  disease  in  animals 
to  which  they  were  transferred.  "  The  optimum  time  for  the 
inoculation  was  from  five  to  ten  days  after  the  sensitizing 
dose,"  they  report. 

They  detail  one  experiment  on  one  animal  with  the  tuber- 
cle bacillus  which  I  shall  quote  : 

"  A  rabbit  of  2,500  grams  was  inoculated  with  .05  milligram  of  human 
tubercle   bacilli,  fourteen  days  after  sensitization  with    50   milligrams   of 


STUDIES    IN    IMMUNITY    TO    TUBERCULOSIS.  37 

killed  pulverized  tubercle  bacilli  (Meister,  Lucius,  and  Briining).  The 
rabbit  died  in  one  week,  having  the  post-mortem  appearance  of  acute  mili- 
ary tuberculosis,  the  only  difference  being  large  necrotic  areas  in  the 
spleen. 

"The  control  rabbit  of  2,300  grams  received  only  a  dose  of  .05  milli- 
gram of  the  same  tubercle  culture  as  was  used  for  the  previous  animal. 
Six  months  later  it  was  alive  and  well,  and  after  killing  it  a  post-mortem 
examination  revealed  no  signs  of  tuberculosis." 

Now  the  reports  of  a  number  of  Thiele  and  Embleton's 
experiments  were  at  total  variance  with  what  had  been  our 
experience  at  the  Saranac  I  aboratory.  I  had  often  made 
repeated  inoculations,  spaced  at  various  intervals,  of  the  vari- 
ous saprophytic  acid-fast  bacilli,  and  had  never  noted  any 
effect  on  the  animal  that  would  compare  with  what  Thiele 
and  Embleton  found.  I  had  also  frequently  practised  the 
procedure  outlined  by  them  in  the  above  experiment  with 
tubercle  bacilli  and  had  not  arrived  at  a  similar  result.  It  is 
true  that  my  technic  had  not  been  exactly  similar  to  theirs  : 
my  experiments  had  been  done  in  work  performed  from  very 
different  points  of  view.  But  their  results  were  so  surprising 
in  view  of  what  I  had  already  been  taught  by  my  own  work 
that  I  resolved  to  repeat  systematically  some  of  the  experi- 
ments, using  the  tubercle  bacillus  for  the  infecting  agent.  I 
aimed,  too,  to  reproduce  their  technic  in  its  essential  features. 

I  therefore  outlined  the  following  experiment:  Inocula- 
tion of  guinea-pigs  with  very  large  quantities  of  dead  virulent 
tubercle  bacilli;  re-inoculation  ten  days  afterwards  with  large 
quantities  of  living  tubercle  bacilli  of  low  virulence  ;  compare 
the  effects  on  the  re-inoculated  animals  as  against  those  on 
controls. 

1.  Material  used  for  primary  inoculation.  —  For  my  pri- 
mary inoculation  I  desired  tubercle  bacilli  that  were  dead 
and  which  nevertheless  retained  their  integrity  as  much  as 
possible  —  which,  in  other  words,  had  not  been  altered  by 
heating  or  by  chemicals.  I  therefore  selected  the  following 
material :  In  the  Saranac  Laboratory  we  are  constantly  col- 
lecting  the   bacilli   from   broth   cultures,  washing   them,  and 


38  KRAUSE. 

drying  them  in  the  dark  in  vacuo  over  sulphuric  acid.  As 
these  accumulate  we  put  them  aside  in  the  dark  in  a  dried 
state.  They  have  been  subjected  to  no  other  agencies  than 
washing  and  drying  and  their  original  constitution  has  pre- 
sumably not  been  altered.  At  the  time  at  which  I  began 
this  experiment  (June,  191 5),  we  had  in  one  bottle  a  collec- 
tion of  originally  virulent  H37  strain  which  had  been  washed, 
dried,  and  assembled  between  January,  1914,  and  March, 
19 14  At  this  distance,  fifteen  to  seventeen  months,  they 
were  presumably  dead  and  I  resolved  to  use  these  for  my 
sensitizing  inoculation. 

2.  Primary  inoculation.  —  June  21.  Two  grams  of  the 
dried  H37  tubercle  bacilli  were  ground  by  hand  in  an  agate 
mortar  for  one  and  one-half  hours.  Twenty-five  cubic  centi- 
meters of  physiological  salt  solution  were  then  added  and 
the  bacilli  were  carefully  emulsified.  One  cubic  centimeter 
of  the  emulsion  therefore  represented  eighty  milligrams  of 
the  dried  bacilli. 

On  the  same  day  twenty  guinea-pigs  were  inoculated  sub- 
cutaneously  in  the  right  groin  as  follows :  They  were 
divided  into  two  lots  of  ten  each:  lot  No.  1,  Nos.  1  to  10, 
and  lot  No.  2,  Nos.  n  to  20.  Nos.  I  and  2  and  10  to  13 
received  .5  cubic  centimeter  each  (40  mg.  dried  bacilli)  ; 
Nos.  3  to  5  and  14  and  15,  .25  cubic  centimeter  each  (20 
mg.  dried  bacilli)  ;  Nos.  6  to  10  and  16  to  20,  .1  cubic  centi- 
meter each  (8  mg.  dried  bacilli). 

3.  Re-inoculation.  —  July  I.  Ten  days  after  primary 
inoculation.  Re-inoculation  was  made  with  Ri,  described 
in  the  foregoing  pages.  A  heavy,  milky,  non-centrifugalized 
emulsion  of  Ri  was  prepared.  Of  the  above  animals,  Nos. 
1  to  10  were  inoculated  subcutaneously,  each  with  .25  cubic 
centimeter  of  the  Ri  emulsion  in  the  left  groin.  Nos.  11 
to  20  were  not  re-inoculated  with  Ri  emulsion,  but  were 
left  to  control  the  effects  of  the  original,  primary  inoculation 
with  old,  dried  H37.  At  the  same  time,  five  new  animals 
Nos.  21    to   25,   which  were  normal,  untreated  animals,  were 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  39 

inoculated  like  Nos.  1  to  10,  each  with  .25  cubic  centimeter 
of  the  Ri  emulsion  to  control  the  effects  produced  by  Ri. 
We  therefore  had  three  sets  of  animals : 

Set  1  :      Nos.  1  to  10.     Inoculated  June  21  with  old,  dried 

H37  and  re-inoculated  July  I 
with  living  Ri. 

Set  2:      Nos.    11    to   20.      Inoculated    June    21    with    old, 

dried  H37. 

Set  3:      Nos.  21  to  25.      Inoculated  July  I  with  living  Ri. 

With  Set  2  we  could  study  the  effects  of  a  single  dried 
H37  inoculation;  with  Set  3,  those  of  a  single  living  Ri 
inoculation ;  and  we  could  compare  both  with  those  pro- 
duced in  the  doubly  inoculated  animals  of  Set  I. 

4.  Results.  —  August  9.  Forty  days  after  inoculation 
with  Ri.  All  the  animals,  Nos.  1  to  25,  were  killed  and' 
autopsied. 

Nos.  1  to  10:  All  have  large  caseous  glands  and  abscesses 
in  the  right  groin  (site  of  dried  H37  inoculation)  and  not 
quite  such  large,  caseating  glands  in  the  left  groin  (Ri 
inoculation).  The  iliac  glands  are  considerably  enlarged 
(right  and  left)  and  have  caseous  spots  in  all  of  them.  In 
no  instance  has  the  disease  got  beyond  the  iliac  glands ; 
there  are  no  visible  lesions  in  the  spleens,  livers,  or  lungs  of 
any  of  the  animals. 

Nos.  11  to  20 :  Those  animals  that  had  only  an  inocula- 
tion of  old,  dried  H37  on  June  21. 

In  general,  the  effects  are  the  same  as  those  in  Nos.  1  to 
10,  except  that  there  is  no  enlargement  or  involvement  of 
the  left  superficial  and  deep  inguinal  glands.  The  iliac 
glands  on  both  sides  are  enlarged.  In  No.  14  the  spleen  is 
slightly  enlarged  with  numerous  small,  pin-head,  raised, 
grayish-yellow  nodules  resembling  tubercles.  In  No.  15  the 
spleen  is  slightly  enlarged  and  shows  one  raised,  yellowish 
nodule,  five  millimeters  in  diameter.  In  No.  17  the  spleen 
is  almost  of  normal  size,  but  has  one  small,  raised,  yellowish- 
nodule,  two  millimeters  in  diameter.     The  spleens  of  all  the 


40  KRAUSE. 

other  animals  seem  normal.  The  livers  and  lungs  of  all  the 
animals  are  apparently  normal. 

Nos.  21  to  25  :  Those  animals  that  had  only  an  inocula- 
tion of  living  Ri  on  July  1. 

There  is  involvement  of  the  superficial  and  deep  inguinal 
and  iliac  glands  of  the  left  side  in  all.  All  spleens,  livers, 
and  lungs  seem  normal. 

5.  Discussion  of  Experiment  4.  —  It  will  be  seen  that 
those  animals  of  Set  I  that  were  treated  according  to  Thiele 
and  Embleton's  method,  i.e.,  those  that  had  received  a  double 
inoculation  during  a  ten-day  interval,  did  not  differ  materially 
from  those  of  Set  3  that  had  only  the  Ri  or  control  inocu- 
lation. But  in  Set  2  which  had  only  the  old,  dried  H37 
inoculation,  and  which  was  used  to  control  this  inoculation, 
several  interesting  and  surprising  results  occurred.  In  Nos. 
14,  15,  and  17  the  spleens  were  found  to  be  involved  with 
what  looked  like  tubercle.  These  nodules  were  accordingly 
enucleated  and  ground  up,  each  in  a  separate  mortar. 
Smears  were  prepared  from  them  and  stained.  After 
prolonged  search,  one  acid-fast  bacillus  was  found  in  the 
smear  made  from  the  spleen  of  No.  15  ;  none  was  found  in 
those  from  the  spleens  of  Nos.  14  and  17.  Each  emulsion 
of  spleen  juice  was  therefore  re-inoculated  separately  into  a 
normal  guinea-pig  on  August  9.  On  September  25  these 
three  guinea-pigs  were  killed  and  autopsied,  and  all  showed 
a  tuberculous  involvement  that  corresponded  with  what  we 
usually  obtain  when  we  inoculate  living  H37  under  ordinary 
conditions.  In  all  three  diagnostic  animals  the  spleens  were 
considerably  enlarged  and  contained  caseous  nodules.  It 
will  be  remembered  that  Nos.  14,  15,  and  17  were  inoculated 
only  with  old  H37  bacilli,  which  had  been  thoroughly  dried 
from  fifteen  to  seventeen  months  before.  It  was  astonishing 
to  us  that  under  such  conditions  these  microorganisms 
should  maintain  their  viability  and  their  parasitism  Yet 
such  was  undoubtedly  the  case.  Transference  of  the  spleen 
nodules  of  Nos.  14,  15,  and  17  showed  that  under  the  above 
mentioned  conditions  H37  had   lost  none  of  its  virulence  as 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  4 1 

we  had  learned  to  know  it.  It  would  appear  that  in  the 
great  mass  of  dried  organisms  that  had  been  preserved  over 
all  this  time  a  few  out  of  countless  numbers,  a  few  perhaps 
far  in  the  interior  of  large  clumps,  had  remained  alive  during 
fifteen  to  seventeen  months. 

The  net  result  of  the  experiment,  however,  carefully  con- 
trolled as  regards  the  infectiousness  of  the  two  types  of 
tubercle  bacilli  used,  failed  to  substantiate  Thiele  and 
Embleton's  view  that  a  sensitization  of  from  five  to  fifteen 
days  can  so  diminish  the  resistance  of  the  animal  organism 
that  the  parasitism  of  a  bacillary  invader  may  be  increased 
thereby.  The  result,  too,  is  more  or  less  in  harmony  with 
those  of  the  first  three  experiments  detailed  in  this  paper. 

GENERAL  DISCUSSION  AND  SUMMARY. 
The  foregoing  work  is  merely  a  first  attempt  to  study  the 
anaphylactic  state  as  it  is  related  to  resistance  to  infection  by 
the  tubercle  bacillus  and  to  the  extension  of  tuberculous 
disease.  Under  the  conditions  of  the  experiments,  especially 
experiments  Nos.  1  and  2,  no  relationship  has  been  con- 
clusively proved.  Yet  other  methods  of  attack  remain  and 
it  is  to  be  hoped  that  these  will  be  applied.  It  is  certainly  a 
plausible  view  that  under  the  influence  of  the  continuous 
absorption  of  bacillary  protein  and  intoxication  thereby, 
resistance  may  be  so  lowered  that  bacillary  invasion  is  a 
comparatively  easy  matter.  After  watching  the  course  of 
long-continued  febrile  phthisis  one  gets  this  impression. 
The  experiments  herein  outlined  were  concerned  with  the 
influence  on  resistance  of  a  single,  marked,  anaphylactic 
intoxication.  In  Experiment  1  it  may  have  had  an  effect, 
but  this  was  so  obscure  that  no  generalization  could  be 
drawn  therefrom.  In  Experiment  2  it  had  apparently  no 
effect.  But  here  we  must  remember  that  the  existing 
disease  may  perhaps  have  been  so  well  localized  and  walled 
off  that  no  bacilli  were  mobilized  and  none  were  free  to 
exert  their  influence  on  freshly  anaphylactized  soil.  If,  after 
infection  was  once  established,  an  animal  could  be  kept  in  a 
more  or  less  continuously  intoxicated  condition  {i.e.,  anaphy- 
laxis intoxication)  perhaps  the  result  would  be  different. 


42  KRAUSE. 

Under  the  experimental  condition  above  outlined  we  are 
justified  in  concluding  : 

i.  Anaphylactic  shock,  experienced  by  guinea-pigs  a 
short  time  before  infection  with  tubercle  bacilli  of  low  viru- 
lence, did  not  reduce  their  resistance  to  such  an  extent  that 
the  parasitism  of  the  particular  microorganism  concerned 
was  markedly  increased,  although  there  were  suggestions  that 
the  extent  of  disease  was  probably  increased. 

2.  If  tuberculous  disease  of  low  grade  is  once  established 
in  guinea-pigs  a  single  attack  of  anaphylaxis  does  not  bring 
about  conditions  that  favor  the  extension  of  the  disease. 

3.  Anaphylactic  shock  suffered  just  before  the  inoculation 
of  a  non-pathogenic  acid-fast  organism,  the  Mist  Bacillus, 
does  not  lay  the  body  open  to  progressive  invasion  by  this 
germ. 

4.  No  success  attended  the  efforts  to  enhance  the  viru- 
lence of  a  strain  of  the  tubercle  bacillus  (Ri)  when  the 
method  of  Thiele  and  Embleton  was  followed. 

5.  Evidence  is  herewith  submitted  that  tubercle  bacilli 
can  preserve  their  viability  and  their  original  virulence  after 
being  kept  in  a  dried  state  for  as  long  a  time  as  from  fifteen 
to  seventeen  months. 

[This  work  was  made  possible  through  the  generosity  of  Mrs.  A.  A. 
Anderson  of  New  York  City.] 

BIBLIOGRAPHY. 

1.  Vagedes.     Zeitschr.  f.  Hyg.,  1898,  xxviii,  276. 

2.  Krompecher  and  Zimmermann.  Centralbl.  f.  Bakter.,  Orig.,  1903, 
xxxiii,  580. 

3.  Frankel  and  Baumann.     Zeitschr.  f.  Hyg.,  1906,  liv. 

4.  Moller.     Ibid.,  1906,  lv,  506. 

5.  Burnet.     Ann.  de  l'lnst.  Past.,  1912,  xxvi,  868. 

6.  Trudeau.     Amer.  Jour.  Med.  Sci.,  1SS7,  No-  I§7>  New  Series,  11S. 

7.  Murphy  and  Ellis.     Journ.  Exp.  Med.,  1914,  xx,  397. 

8.  Arloing.     Compt.  Rend.  Soc.  de  Biol.,  1905,  58,  261. 

9.  Lewis  and  Margot.     Journ.  Exp.  Med.,  1914,  xix,  187. 

10.  Krause.     Journ.  Med.  Research,  191 1,  xxiv,  399. 

11.  Thiele  and  Embleton.  Zeitschr  f.  Imm.  'frschung,  Orig.,  1913, 
xix,  643. 


Studies  in  Immunity  to  Tuberculosis.* 
concerning  the  general  tuberculin  reaction. 

A.  K.  Krause,  M.D. 
{From  the  Saranac  Laboratory  for  the  Study  of  Tuberculosis. ,) 

Several  years  ago  in  a  discussion  of  the  probable  mechan- 
ism of  the  general  tuberculin  reaction  I1  took  the  ground 
that  this  reaction  is  at  bottom  dependent  upon  a  focal  reac- 
tion. This  was  a  point  of  view  that  had  been  advanced 
before  and  has  always  had  a  number  of  adherents.  In  elab- 
orating it  I  sought  to  explain  the  well-known  symptoms  of 
intoxication  that  mark  the  general  reaction  and  suggested 
that  these  were  due  to  the  reaction  of  some  absorbed  focal 
products  which  exerted  their  effect  upon  an  organism  that 
had  already  become  hypersensitive  to  them  by  reason  of 
previous  focal  absorption.  We  could  then  comprehend  the 
series  of  events  that  make  up  this  exceedingly  complicated 
reaction  as  follows  :  The  antigen  (tuberculin)  injected  into 
the  body  is  absorbed  and  in  due  time  reaches  the  tuberculous 
focus,  with  which  it  will  react.  This  focal  reaction  is  mani- 
fested anatomically  by  exudation  or  inflammation,  the  sever- 
ity of  which  is  dependent  upon  a  combination  of  at  least  two 
factors;  first,  the  accessibility  of  the  focus,  that  is,  the  ease 
with  which  the  antigen  can  reach  it,  a  condition  which  will 
vary  with  the  degree  of  fibrous  investment  of  the  focus  ;  and 
second,  the  amount  of  tuberculin  which  gets  to  the  focus. 
The  acute  focal  inflammation  favors  the  absorption  into  the 
blood  or  lymph  channels  of  focal  products,  which  is  more 
rapid  and  quantitatively  greater  than  before  the  reaction. 
This  sudden  absorption  of  comparatively  large  amounts  of 
focal  material  reacts  on  an  already  sensitized  body,  produc- 
ing an  anaphylactic  intoxication,  the  symptoms  of  which  we 
term  the  general  reaction. 

Since  then  I  have  had  no  reason  to  change  my  belief  that  the 

*  Received  for  publication  June  10,  1916. 
(43) 


44  KRAUSE. 

absorption  of  focal  products  brings  about  the  general  reac- 
tion. But  the  results  of  a  series  of  experiments  undertaken 
to  prove  the  validity  of  the  above  hypothesis  have  led  me  to 
modify  my  view  that  the  symptoms  are  anaphylactic  in 
origin. 

My  first  experiments  were  made  with  the  idea  of  determin- 
ing whether  the  reacting  focus  contains  a  primary  toxin, 
and,  if  it  does,  whether  this. toxin  is  present  during  the  late 
stages  of  reaction  in  larger  amounts  than  during  the  earlier 
phases.  Guinea-pigs  with  advanced  tuberculosis  received 
intraperitoneal  injections  of  varying  quantities  of  a  water 
extract  of  dried,  pulverized  tubercle  bacilli.  As  they  fell 
sick  they  were  killed  at  various  times,  some  at  the  beginning 
of  symptoms,  some  after  illness  was  well  established,  and 
others  when  moribund.  Their  tuberculous  organs  showing 
the  familiar  focal  reaction  of  inflammation  were  recovered 
and  ground  in  mortars  or  their  pulp  squeezed  out  in  a  press. 
The  pulp  was  taken  up  in  physiological  salt  solution  and  the 
resultant  suspension  was  centrifugated.  The  supernatant 
fluid  was  then  used  for  intravenous  injection  into  normal 
guinea-pigs.  The  result  was  uniformly  the  same.  All 
animals  thus  injected  became  very  acutely  ill,  and  generally 
died  in  from  one  to  five  minutes.  The  solution  of  tubercu- 
lous organ  pulp  was  very  dilute;  the  lethal  dosage  com- 
paratively small,  as  low  as  one  cubic  centimeter.  Quantitative 
experiments  failed  to  show  that  the  organs  of  moribund 
animals  with  intense  hemorrhagic  focal  reactions  were  any 
more  toxic  than  those  with  beginning  reaction,  in  which  the 
focal  inflammation  was  not  visible  to  the  naked  eye. 

I  was  next  led  to  test  the  toxicity  of  ordinary,  non-react- 
ing foci.  Tuberculous  organs  of  guinea-pigs  were  treated 
in  essentially  the  same  manner  as  outlined  above  and  the 
solutions  were  injected  intravenously  into  normal  guinea- 
pigs.  Such  animals  exhibited  the  same  symptoms  and  died 
as  acutely  as  though  they  had  received  the  products  of  the 
reacting  focus.  Sometimes,  if  the  dose  was  very  small  they 
would  almost  immediately  become  very  ill  and  then  gradually 
recover.     Sometimes  the  supernatant  diluted  tuberculous  lung 


STUDIES   IN    IMMUNITY   TO   TUBERCULOSIS.  45 

juice  or  spleen  juice  was  divided  into  two  portions,  one  of 
which  was  passed  through  Berkefeld  bougie  or  cotton  or 
paper.  The  filtrate  was  apparently  harmless,  while  the 
unfiltered  fluid  was  highly  poisonous.  If  the  material  was 
injected  intraperitoneally  instead  of  intravenously  the  animal 
suffered  no  apparent  discomfort,  but  would  of  course 
develop  tuberculosis  after  the  proper  incubation  interval. 
The  net  result  of  this  series  of  experiments  was  that  I  could 
detect  no  difference  between  the  effects  of  reacting  focal 
products  and  those  of  non-reacting  foci. 

I  then  compared  the  foregoing  results  with  those  obtained 
from  the  injections  of  similar  preparations  of  normal  organs 
of  healthy  guinea-pigs.  It  was  found  that  the  juice  of 
normal  organs  produced  exactly  the  same  effects.  Wasser- 
mann  and  Keyser 2  had  reported  that  the  blood  serum  of 
normal  guinea-pigs  was  toxic  for  other  normal  guinea-pigs,  if 
injected  intravenously  into  the  latter.  In  common  with 
many  other  authors  I  was  unable  to  confirm  their  observa- 
tions. I  usually  killed  my  normal  guinea-pigs  by  bleeding 
them  and  used  the  serum  thus  obtained  for  intravenous 
injections  on  normal  hosts.  So  far  as  I  could  determine  such 
injections  never  produced  the  least  harmful  effect  on  the 
recipient,  even  though  the  latter  received  ten  cubic  centi- 
meters and  more.  The  juice  of  organs  of  the  same  donors 
was,  however,  uniformly  toxic.  It  was  therefore  plain  that 
the  organs  were  not  toxic  because  of  any  blood  from  which 
they  had  not  been  freed.  Water  extracts  of  dried  spleens 
and  lungs  were  also  harmless.  These  organs  were  dried  at 
500  C.  for  twenty-four  hours,  then  powdered  and  extracted 
in  distilled  water  at  500  C.  for  twenty-four  hours.  The  emul- 
sion was  made  isotonic  and  centrifugalized.  The  resultant 
supernatant  fluid  produced  no  symptoms  when  injected 
intravenously  into  guinea-pigs. 

From  these  experiments  it  would  seem  reasonable  to  con- 
clude that  any  tissue  products  if  thrown  rapidly  into  the  cir- 
culation in  unusual  amounts  will  intoxicate  an  animal.  In 
other  words,  one's  own  tissues,  whether  normal  or  patholog- 
ical, may  conceivably  poison  one  if  they  get  into  the  blood 


46  KRAUSE. 

stream  in  excess.  Therefore,  if  the  focal  reaction,  following 
a  tuberculin  injection,  establishes  a  better  circulatory  give 
and  take  between  the  tuberculous  focus  and  the  animal 
organism,  and,  as  it  were,  "  unlocks"  or  "opens"  the  focus, 
the  resultant  symptoms  of  a  general  reaction  may  be  due 
merely  to  substances  that  are  primarily  toxic  for  the  organ- 
ism. The  same  absorption  bringing  about  symptoms  of 
illness  will  occur  whenever  the  conditions  for  focal  absorp- 
tion are  enhanced,  whether  it  be  the  more  or  less  continuous 
absorption  from  a  poorly  invested  focus  or  the  occasional 
absorption  following  physical  strain,  intercurrent  disease,  or 
drug  action,  any  of  which  may  produce  circulatory  condi- 
tions about  the  focus  that  favor  increased  absorption. 

It  is  interesting  that  in  a  recent  paper,  "  The  Cellular 
Factor  in  Infectious  Diseases,"  Riesman3  puts  forward  a  sim- 
ilar hypothesis  to  explain  the  toxemia  in  certain  conditions 
that  are  remarkable  for  cellular  proliferation  and  exudation, 
such  as  miliary  tuberculosis,  carcinomatosis,  pneumonia,  etc. 
He  conceives  that  in  these  and  similar  other  "  productive  " 
diseases,  the  proliferated  cells  which  are  present  in  enormous 
numbers  undergo  parenteral  digestion  and  in  consequence 
throw  "  into  the  blood  products  that  if  not  qualitatively  are 
at  least  quantitatively  abnormal,"  and  which  may  give  rise  to 
a  large  share  of  the  symptoms  of  such  diseases.  The  fore- 
going work  furnishes  some  experimental  basis  for  Riesman's 
view. 

While  it  would  seem  from  the  above  experiments  that 
normal  tissues,  introduced  parenterally,  are  toxic  for  the 
organism,  it  is  difficult  to  imagine  how  normal  tissues  could 
enter  the  circulation  if  an  individual  is  in  perfect  health. 
But  given  a  fracture  of  bone,  an  extensive  burn,  or  a  focus 
of  disease,  and  conditions  are  at  once  established  that  favor 
the  absorption  of  cellular  elements  to  which  the  body  may 
react  with  symptoms  of  illness,  the  severity  of  which  would 
be  proportional  to  the  amount  of  material  taken  up  in  a 
given  time. 


STUDIES   IN   IMMUNITY   TO   TUBERCULOSIS.  47 

SUMMARY. 

1.  An  extract  of  an  animal's  own  normal  tissues  if  intro- 
duced rapidly  into  its  circulation  is  toxic. 

2.  The  products  of  tuberculous  foci  are  primarily  toxic 
if  received  into  the  blood  stream  (or,  perhaps,  lymph 
stream). 

3.  It  is  suggested  that  the  symptoms  of  the  general 
tuberculin  reaction  are  due  to  the  primary  toxicity  of  focal 
products,  the  absorption  of  which  is  favored  by  the  focal 
reaction  that  results  from  the  injection  of  tuberculin. 

Record  of  Experiments. 
A.     Injections  of  Tuberculous  Tissue  Juices : 

1.  February  21 :  Five  guinea-pigs,  inoculated  intraperitoneally  on 
January  27th  with  tubercle  bacilli,  H39,  were  bled  to  death  by  cardio- 
puncture.  In  every  case  the  omentum  was  rolled  up,  nodular  and  caseous. 
All  had  moderate  miliary  tuberculosis  of  the  spleen.  One  liver  showed  a 
number  of  pinhead,  caseous  tubercles.  There  was  no  tuberculosis  of  the 
lungs  in  any.  The  omenta  and  spleens  of  the  five  animals  and  a  small 
piece  of  tuberculous  liver  were  ground  together  in  a  meat  chopper,  mixed 
with  15  cubic  centimeters  of  physiological  salt  solution,  and  pressed  out 
with  an  oil  press. 

(a)  Guinea-pig  (550  gms.),  normal,  injected  intravenously  with  5  cubic 
centimeters  of  the  tuberculous  organ  juice.  It  immediately  went  into 
collapse  and  died  in  one  and  one-half  minutes  Autopsy  :  No  macro- 
scopic lesions 

(b)  Guinea-pig  (650  gms.),  normal,  injected  intravenously  with  2.5 
cubic  centimeters.  Lively  for  one  or  two  minutes,  then  collapsed  and 
death  in  three  minutes.     Autopsy:     No  macroscopic  lesions. 

The  material  was  then  centrifugalized  and  the  supernatant  liquid  used 
for  the  next  injection. 

(c)  Guinea-pig  (350  gms.),  normal,  injected  intravenously  with  1  cubic 
centimeter  of  the  supernatant  liquid.  Lively  for  one  minute,  then  sudden 
collapse  and  convulsions,  with  death  in  three  minutes.  Autopsy:  Lungs 
pale  and  slightly  insufflated,  few  epicardial  hemorrhages. 

The  supernatant  fluid  was  then  filtered  through  paper  and  the  filtrate 
used  for  the  next  injection. 

(d)  Guinea-pig  (270  gms.),  normal,  injected  intravenously  with  .75 
cubic  centimeter  of  the  filtrate.     No  symptoms. 

2.  October  5:  Three  tuberculous  guinea-pigs,  inoculated  September 
9th  with  tubercle  bacilli,  H37,  bled  to  death.  All  spleens  had  diffuse, 
caseating  miliary  tuberculosis.     The  spleens  were  crushed  with  sand  in  a 


48  KRAUSE. 

mortar,  mixed  with  10  cubic  centimeters  of  distilled  water,  and  stood  in 
the  ice-chest  for  twenty-four  hours.  The  emulsion  was  then  filtered 
through  cotton.     Total  filtrate  equals  7  cubic  centimeters. 

October  6:  Guinea-pig  (650  gms.),  normal,  injected  intravenously 
with  6  of  filtrate.     No  symptoms. 

B.     Injections  of  Non-tuberculous  Tissue  Juices: 

1.  February  23:  Five  normal  guinea-pigs  were  bled  to  death.  Their 
omenta  and  spleens  and  a  small  piece  of  liver  of  one  pig  were  ground 
together  in  a  meat  chopper,  mixed  with  10  cubic  centimeters  of  physio- 
logical salt  solution,  and  pressed  out  in  an  oil  press. 

February  24:  (a)  Guinea-pig  (500  gms.),  normal,  injected  intrave- 
nously with  4  cubic  centimeters  of  normal  organ  juice.  Lively  for  two 
minutes,  then  restless,  urinating,  bucking,  falls  over,  convulsions,  marked 
inspiratory  spasms,  death  in  four  minutes.  Autopsy:  No  macroscopic 
lesions. 

(b)  Guinea-pig  (530  gms.),  normal,  injected  intravenously  with  2.5 
cubic  centimeters.  Lively  for  two  minutes,  then  uneasy,  urinating, 
coughing,  bucking,  falls  over,  convulsions,  unconscious,  death  in  four 
minutes.     Autopsy  :     Lungs  pale,  slightly  blown  out. 

The  juice  was  then  centrifugalized  and  the  supernatant  fluid  filtered 
through  paper. 

(c)  Guinea-pig  (500  gms.),  normal,  injected  intravenously  with  5 
cubic  centimeters  of  filtrate.     No  symptoms . 

2.  December  9:  Three  non-tuberculous  guinea-pigs  bled  to  death. 
The  liver  tissue  from  three  animals  and  the  lungs  and  spleen  from  two 
were  taken  and  treated  separately.  Moist  weights;  liver,  20  grams; 
lungs,  7.5  grams;  spleen,  1  gram.  All  the  organs  were  crushed  in 
mortars  with  sand  and  mixed  with  physiological  salt  solution,  10  cubic 
centimeters  to  liver  pulp,  10  cubic  centimeters  to  lung  pulp,  and  2  cubic 
centimeters  to  spleen  pulp.  The  different  emulsions  were  centrifugalized 
and  the  supernatant  fluids  pipetted  off.  These  fluids  were  even  in  appear- 
ance and  contained  no  macroscopic  particles. 

(a)  Guinea-pig  (270  gms.),  normal,  injected  intravenously  with  1  cubic 
centimeter  of  supernatant  fluid  from  spleen  juice.  Animal  lively  for  one 
minute,  then  twitching,  convulsions,  opisthotonus,  rapid  respiration. 
Animal  gradually  recovered. 

(b)  Guinea-pig  (300  gms.),  normal,  injected  intravenously  with  3 
cubic  centimeters  of  lung  juice  fluid.  No  symptoms  for  one  minute,  then 
spasmodic  movements,  convulsions,  death  in  four  minutes.  Respiratory 
picture  at  end  very  much  like  that  of  anaphylaxis.  Autopsy:  Lungs 
resemble  the  insufflated  lungs  of  anaphylaxis,  but  are  not  so  pale  and  not 
so  distended  ;  no  epicardial  hemorrhages. 

The  liver  juice  fluid  was  filtered  through  ordinary  filter  paper,  hard 
filter  paper  and  cotton,  successively,  and  the  filtrate  used  in  the  following 
injection : 


STUDIES    IN    IMMUNITY   TO    TUBERCULOSIS.  49 

(c)  Guinea-pig  (220  gms.),  normal,  injected  intravenously  with  2  cubic 
centimeters  of  filtrate.  Same  symptoms  as  foregoing  animal,  b  ;  death 
in  two  minutes.     Autopsy:  Lungs  moderately  distended.     No  lesions. 

This  is  the  only  instance  in  which  a  filtrate  of  tissue  juice  produced 
intoxication. 

(d)  Guinea-pig  (260  gms.),  normal,  injected  intravenously  with  5  cubic 
centimeters  of  mixed  blood  serum  from  the  same  animals  from  which  the 
organs  were  taken.     No  symptoms. 

3.  December  15:  Material  consisted  of  lungs  from  five  non-tubercu- 
lous guinea-pigs,  four  of  which  had  been  bled  to  death.  Total  moist 
weight  is  17  grams.  Ground  in  mortar  with  sand  and  mixed  with  25 
cubic  centimeters  of  physiological  salt  solution.  Stood  in  ice-box  over 
night.  The  next  morning  centrifugalized  and  the  supernatant  fluid 
divided  into  two  portions,  one  to  undergo  Berkefeld  filtration.  The  blood 
serum  was  removed  from  the  clots  one  hour  after  bleeding,  incubated  at 
2,7°  C.  for  one  hour  and  fifteen  minutes,  then  stood  at  room  temperature 
for  twenty-four  hours. 

December  16:  (a)  Guinea-pig  (300  gms.),  normal,  injected  intra- 
venously with  7.5  cubic   centimeters  of  blood  serum.     No  symptoms. 

(b)  Guinea  pig  (300  gms),  normal,  injected  intravenously  with  less 
than  1  cubic  centimeter  of  unfiltered  lung  fluid.  Collapse  coming  on  one 
minute  after  injection.     Slow  recovery. 

(c)  Guinea-pig  (300  gms.),  normal,  injected  intravenously  with  2  cubic 
centimeters  unfiltered  lung  fluid.  Same  symptoms  as  foregoing  animal, 
b.     Slow  recovery. 

(d)  Guinea-pig  (220  gms.),  normal,  injected  intravenously  with  1.2 
cubic  centimeters  filtrate.     No  symptoms. 

4.  December  19 :  Three  non-tuberculous  guinea-pigs  were  bled  to 
death.  The  sera  of  the  three  animals  were  mixed  together  and  part  of 
the  mixture  was  incubated  one  and  one-half  hours  at  370  C.  and  stood  at 
room  temperature  for  twenty-four  hours.  The  other  part  was  not  incu- 
bated and  stood  in  the  ice-box  for  twenty-four  hours. 

The  livers  were  crushed  with  sand  in  a  mortar  and  mixed  with  physio- 
logical salt  solution.  The  mixture  was  centrifugated  and  the  supernatant 
fluid  used. 

The  lungs  and  spleen  were  dried  on  a  water  bath  at  500  C.  for  twenty- 
four  hours.  They  were  then  powdered  and  extracted  in  distilled  water  for 
twenty-four  hours  at  500  C.  A  few  cubic  centimeters  of  physiological  salt 
solution  were  then  added  and  the  emulsion  was  centrifugated.  The  super- 
natant fluid  was  used  for  injection. 

(a)  Guinea-pig  (250  gms.),  normal,  injected  intravenously  with  2.5 
cubic  centimeters  of  liver  juice  fluid.  Collapse  and  death  in  two  minutes. 
Autopsy:  Lungs  collapsed,  no  lesions. 

December  20:  (b)  Guinea-pig  (350  gms),  normal,  injected  intra- 
venously with  10  cubic  centimeters  of  the  incubated  serum  mentioned 
above.     No  symptoms. 


50  KRAUSE. 

(c)  Guinea-pig  (300  gms.),  normal,  injected  intravenously  with  8  cubic 
centimeters  of  the  non-incubated  serum.     No  symptoms. 

December  21:  (d)  Guinea-pig  (270  gms),  normal,  injected  intra- 
venously with  4  cubic  centimeters  of  the  extract  of  dried  spleens.  No 
symptoms. 

.(e)  Guinea-pig  (280  gms.),  normal,  injected  intravenously  with  3  cubic 
centimeters  of  the  extract  of  dried  lungs.     No  symptoms. 

Discussion.  —  Kraus  and  Volk4  have  reported  that  fil- 
trates of  extracts  of  tuberculous  focal  material  are  primarily- 
toxic  for  guinea-pigs,  while  those  of  normal  organs  are  not. 
The  above  experiments  fail  to  confirm  their  observations  as 
to  the  effects  of  tuberculous  organ  filtrates.  The  filtrates  of 
material  from  both  tuberculous  and  normal  organs  are 
usually  non-poisonous,  whether  the  filtration  is  done  through 
a  Berkefeld  filter,  paper,  or  cotton.  The  even  appearance 
of  the  supernatant  fluids  and  their  freedom  from  visible  parti- 
cles dispels  the  idea  that  the  symptoms  are  caused  by 
cerebral  or  pulmonary  emboli.  The  harmlessness  of  the 
dried  tissue  extracts  makes  it  seem  as  though  the  symptoms 
are  not  due  to  protein  intoxication.  It  would  appear  that 
the  cellular  elements  carry  some  intoxicating  substance  which 
exerts  its  action  when  large  enough  quantities  of  cells  or  of 
material  from  disintegrated  cells  are  rapidly  thrown  into  the 
circulation. 

[This  work  was  made  possible  through  the  generosity  of  Mrs.  A.  A. 
Anderson  of  New  York  City.] 

BIBLIOGRAPHY. 

1.  Krause,  A.  K.  Johns  Hopkins  Hospital  Bulletin,  Special  Tubercu- 
losis Number,  1911,  xxii,  250-258. 

2.  Wassermann,  M.,  and  Keyser,  F.     Fol.  Serol.,  191 1 ,  vii,  243-251. 

3.  Riesman,  David.     J.  Am.  M.  Ass.,  1915,  lxiv,  649-652. 

4.  Kraus,  R.,  and  Volk.     Wien.  med.  Wchnschr.,  1910,  xxiii,  289-290. 


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